Compounds and anti-tumor nqo1 substrates

ABSTRACT

Compounds of Formula (I) can be selectively lethal toward a variety of different cancer cell types. The compounds are useful for the management, treatment, control or adjunct treatment of diseases, where the selective lethality is beneficial in chemotherapeutic therapy.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/351,861, filed Apr. 14, 2014, issued as U.S. Pat. No. 9,233,950,which is a National Stage filing under 35 U.S.C. §371 ofPCT/US2012/059988, filed Oct. 12, 2012, which application claimspriority under 35 U.S.C. §119(e) to U.S. Provisional Patent ApplicationNos. 61/547,166 filed Oct. 14, 2011 and 61/662,163 filed Jun. 20, 2012,which applications are incorporated herein by reference in theirentireties.

GOVERNMENT SUPPORT

This invention was made with government support under contract numberCA102792 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

A fundamental challenge in cancer treatment is the discovery ofcompounds that are toxic to cancer cells but not healthy cells. Asalient feature of cancer is rapid and unrestricted cell division. Thevast majority of traditional chemotherapeutics target rapidly dividingcells by disrupting the cell cycle, causing cell death. Because somehealthy tissues require cell division as part of their function,antiproliferative cytotoxins can also kill healthy cells, resulting insevere, dose-limiting side effects. Accordingly, new drugs and newcellular targets must be identified that better differentiate healthyand cancerous cells. These targets may be present in only a smallfraction of cancer patients, making this a personalized strategy totreat cancer.

NAD(P)H quinone oxidoreductase (NQO1, DT diaphorase) is an FAD-dependent2-electron reductase whose primary function is to protect the cell fromcytotoxins, especially quinones. It is a member of the Phase IIdetoxifying enzymes, the expression of which is regulated by NRF-2 andthe antioxidant response element (ARE) in response to electrophilic oroxidative stress. Although generally identified as a cytosolic protein,NQO1 has been identified in subcellular compartments such as themitochondria and nucleus.

Quinone-containing molecules are frequently cytotoxic and harm cellsthrough two mechanisms. Many quinones are conjugate addition acceptorsand readily alkylate nucleophilic species such as DNA and cysteineresidues. Quinones are also substrates for 1-electron reductases, suchas cytochrome P450s, cytochrome bS, xanthine oxidase, and glutathionereductase. Reduction of quinones by these enzymes generates a highlyreactive semiquinone that can damage biomolecules directly, or can beoxidized by dissolved oxygen resulting in the formation of an equivalentof superoxide anion radical and the parent quinone. Thus, 1-electronreduction of quinones can catalytically create reactive oxygen species(ROS) that damage the cell.

By reducing quinones in a 2-electron process, NQO1 bypasses the toxicsemiquinone and forms hydroquinones, which are commonly unreactivetoward oxygen. Hydroquinones are then conjugated with molecules such asglutathione, glucose, or sulfate, and excreted by the cell. However,some hydroquinone-containing molecules are unstable and react withoxygen in two 1-electron oxidations back to the quinone, generating ROS.The relative stability of hydroquinones toward air oxidation cannot bepredicted based on molecular structure and it does not correlate withreduction potential.

NQO1 has attracted much attention as a potential target for thetreatment of cancer because it has been shown to be frequently expressedat much higher levels in tumors relative to adjacent healthy tissue,particularly in the case of lung cancer. In addition, NQO1 activityappears to increase during tumor progression. Other than for lung,breast, and colon tissues, relatively little data on the levels of NQO1in normal tissues has been reported. Whereas low levels of NQO1 arereported in bone marrow and liver cells—two tissues frequently damagedby chemotherapeutics—high levels of NQO1 have been noted in stomach andkidney cells.

The prospect of discovering toxins that are activated, instead ofdeactivated, by NQO1 has attracted researchers for many years. Suchmolecules would turn this normally cytoprotective enzyme into aliability for the cell. Two general classes of molecules have beendiscovered that fit this description: DNA alkylators whoseelectrophilicity is increased after bioreduction, and redox cyclingmolecules that generate ROS catalytically after reduction. Examples ofsuch DNA alkylators include Mitomycin C, EO9, and MeDZQ, and examples ofsuch ROS generators include β-lapachone and streptonigrin, the cytotoxicmechanisms of which each involve NQO1-mediated bioreduction. Theseclasses of molecules are composed almost exclusively ofquinone-containing compounds.

The concentration of β-lap delivered to cells may induce different formsof cell death, with lower concentrations inducing apoptosis and higherconcentrations initiating calcium-dependent necroptosis. In addition toROS generation in RBCs, the poor aqueous solubility of β-lapnecessitates the use of hydroxypropyl-β-cyclodextrin (HPβCD) as asolubility aid, high concentrations of which cause hemolysis of RBCs invitro. To address the issues of compound instability and damage to RBCs,the Boothman and Gao groups have designed a micellar formulation ofβ-lap that demonstrates greatly improved PK properties and efficacy inmurine tumor models (Blanco, Boothman, Gao et al., Cancer Res, 2010, 70,3896).

While personalized medicine strategies have produced life-savinganticancer drugs, they affect only a small percentage of cancerpatients. Because NQO1 levels are highly elevated in a large number ofsolid tumors, a treatment that successfully exploits NQO1 levels couldbenefit a significant fraction of all cancer patients. Despite theextensive efforts expended in discovering and developing NQO1-dependentcytotoxins, none of these compounds are both sufficiently selective forNQO1 and sufficiently stable in vivo to prove whether or not targetingNQO1 overexpression is a viable anticancer strategy. What is needed isevidence that DNQ and its derivatives possess the selectivity andstability required to validate NQO1 as a target for the treatment ofcancer. What is also needed is new compounds and compositions that canselectively inhibit cancer cells and be used in therapeutic cancertherapies.

SUMMARY

The invention provides compounds, compositions and methods to treattumor cells, for example, tumor cells having elevated levels of NQO1. Afirst aspect of the invention thus provides novel DNQ compounds ofFormula (I):

wherein

R₁, R₂, R₃, and R₄ are each independently —H or —X—R;

each X is independently a direct bond or a bridging group, wherein thebridging group is —O—, —S—, —NH—, —C(═O)—, —O—C(═O)—, —C(═O)—O—,—O—C(═O)—O—, or a linker of the formula -W-A-W-, wherein

each W is independently —N(R′)C(═O)—, —C(═O)N(R′)—, —OC(═O)—, —C(═O)O—,—O—, —S—, —S(O)—, —S(O)₂—, —N(R′)—, —C(═O)—, —(CH₂)_(n)— where n is1-10, or a direct bond, wherein each R′ is independently H,(C₁-C₆)alkyl, or a nitrogen protecting group; and

each A is independently (C₁-C₂₀)alkyl, (C₂-C₁₆)alkenyl, (C₂-C₁₆)alkynyl,(C₃-C₈)cycloalkyl, (C₆-C₁₀)aryl, —(OCH₂—CH₂)_(n)— where n is 1 to about20, —C(O)NH(CH₂ 2)_(n)— wherein n is 1 to about 6, —OP(O)(OH)O—,—OP(O)(OH)O(CH₂)_(n), wherein n is 1 to about 6, or (C₁-C₂₀)alkyl,(C₂-C₁₆)alkenyl, (C₂-C₁₆)alkynyl, or —(OCH₂—CH₂)_(n)— interruptedbetween two carbons, or between a carbon and an oxygen, with acycloalkyl, heterocycle, or aryl group;

each R is independently alkyl, alkenyl, alkynyl, heteroalkyl,cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl,(cycloalkyl)alkyl, (heterocycloalkyl)alkyl, (cycloalkyl)heteroalkyl,(heterocycloalkyl)heteroalkyl, aryl, heteroaryl, (aryl)alkyl,(heteroaryl)alkyl, hydrogen, hydroxy, hydroxyalkyl, alkoxy,(alkoxy)alkyl, alkenyloxy, alkynyloxy, (cycloalkyl)alkoxy,heterocycloalkyloxy, amino, alkylamino, aminoalkyl, acylamino,arylamino, sulfonylamino, sulfinylamino, —COR^(x), —COOR^(x),—CONHR^(x), —NHCOR^(x), —NHCOOR^(x), —NHCONHR^(x), —N₃, —CN, —NC, —NCO,—NO₂, —SH, -halo, alkoxycarbonyl, alkylaminocarbonyl, sulfonate,sulfonic acid, alkylsulfonyl, alkylsulfinyl, arylsulfonyl, arylsulfinyl,aminosulfonyl, R^(x)S(O)R^(y)—, R^(x)S(O)₂R^(y)—,—R^(x)C(O)N(R^(x))R^(y)—, R^(x)SO₂N(R^(x))R^(y)—,R^(x)N(R^(x)C(O)R^(y)—, R^(x)N(R^(x))SO₂R^(y)—,R^(x)N(R^(x))C(O)N(R^(x))R^(y)—, carboxaldehyde, acyl, acyloxy, —OPO₃H₂,—OPO₃Z₂ where Z is an inorganic cation, or saccharide; where each R^(x)is independently H, OH, alkyl or aryl, and each R^(y) is independently agroup W;

wherein any alkyl or aryl can be optionally substituted with one or morehydroxy, amine, cyano, nitro, or halo groups;

or a salt or solvate thereof;

provided that when R₁, R₂, and R₃ are methyl, R₄ is not H or methyl; andprovided that when R₁, R₃, and R₄ are methyl, the group —X—R of R₂ isnot —CH₂—OAc. In some embodiments, when R₁, R₃, and R₄ are methyl, the Xgroup of R₂ is not —CH₂—, or the R group of R₂ is not acyloxy.

A second aspect of the invention provides pharmaceutical compositionsthat contain at least one compound of Formula (I) and a pharmaceuticallyacceptable diluent, carrier, or excipient. The invention also providesfor the use of compounds of Formula (I) for the preparation ofpharmaceutical compositions, and the subsequent use of the compositionsin the treatment of patients or subjects. Patients or subjects can bemammals, including humans.

A third aspect of the invention provides methods of treating, killing,or inhibiting the growth tumor cells that have elevated NQO1 levels or atumor having cells that have elevated NQO1 levels, where at least onetumor cell is exposed to therapeutically effective amount of a compoundof Formula (I), a pharmaceutically acceptable salt or solvate thereof,or a pharmaceutically acceptable composition thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Formulas of certain DNQ compounds, according to variousembodiments of the invention.

FIG. 2. Examples of specific DNQ compounds.

FIG. 3. Nude mice were inoculated with A549 cells and tumors wereallowed to establish and grow to 200 mm³. Mice were divided into 6cohorts (4 mice per cohort) and treated on days 1, 3, 5, 7, and 9. Tumorvolumes were measured by caliper.

FIG. 4. X-ray crystal structure of DNQ showing π-stacking in the solidstate.

FIG. 5. Derivatization sites on DNQ according to various embodiments ofthe invention.

FIG. 6. X-ray crystal structure of human NQO1 with inhibitor dicoumarol(left) or with DNQ modeled in using MOE software (right). The FADcofactor is represented in stick form with the adenine moiety at the topof the image and the tricyclin flavin in the active site. Drawings ofthe substrates are included to aid in visualizing the orientation of themolecules in the NQO1 active site.

FIG. 7. Solubility of DNQ derivatives in pH 7.4 PBS. Compounds 4-23,4-32, 4-35, 4-22, 4-34, 4-15, 4-30, 4-37, DNQ, 4-31, 4-14, and 4-8 are“active” compounds (IC₅₀<500 nM), while compounds 4-39, 4-12, 4-9, 4-29,4-11, and 4-41 are generally “inactive” compounds (IC₅₀>500 nM). Theorder of compounds in FIG. 7 is retained for FIGS. 8-11.

FIG. 8. Solubility of active DNQ derivatives in 20% HPβCD. Less activederivatives (4-10, 4-13, 4-28, 4-16, 4-27, 4-26, 4-39, 4-12, 4-9, 4-29,4-11, and 4-41) were not assessed.

FIG. 9. Solubility of DNQ derivatives in DMSO. Compounds 4-25, 4-24,4-23, 4-32, 4-35, 4-22, 4-34, 4-15, 4-30, 4-37, DNQ, 4-31, 4-14, and 4-3are “active” compounds (IC₅₀<500 nM), while compounds 4-28, 4-16, 4-27,4-26, 4-39, 4-12, 4-9, 4-29, 4-11, and 4-41 are generally “inactive”compounds (IC₅₀>500 nM).

FIG. 10. Solubility of DNQ derivatives in dichloromethane. Compounds4-25, 4-24, 4-23, 4-32, 4-35, 4-22, 4-34, 4-15, 4-30, 4-37, DNQ, 4-31,4-14, and 4-8 are “active” compounds (IC₅₀<500 nM), while compounds4-10, 4-13, 4-28, 4-16, 4-27, 4-26, 4-39, 4-12, 4-9, 4-29, 4-11, and4-41 are generally “inactive” compounds (IC₅₀>500 nM).

FIG. 11. Solubility of DNQ derivatives in 33% methanol indichloromethane. Compounds 4-24, 4-23, 4-32, 4-35, 4-22, 4-34, 4-15,4-30, 4-37, DNQ, 4-31, 4-14, and 4-8 are “active” compounds (IC₅₀<500nM), while compounds 4-10, 4-13, 4-28, 4-16, 4-27, 4-26, 4-39, 4-12,4-9, 4-29, 4-11, and 4-41 are generally “inactive” compounds (IC₅₀>500nM).

FIG. 12. Efficacy of DNQ and compound 87 vs, the MDA-MB-231 (breastcancer) cell line that expresses NQO1, the version that does not expressNQO1, and both these cell lines where NQO1 is inhibited by dicumoral.

FIG. 13. Immunoblotting analyses of DNQ-87, DNQ-107 and DNQ-9-251, asdescribed in Example 5, according to one embodiment.

DETAILED DESCRIPTION

Tumor-selectivity remains a challenge for efficacious chemotherapeuticstrategies against cancer. Although the recent development ofβ-lapachone to specifically exploit elevated levels of NAD(P)H:quinoneoxidoreductase 1 (NQO1) in most solid tumors represents a novelchemotherapeutic approach, additional compounds that kill by programmednecrosis at increased potency are needed. This disclosure demonstratesthat deoxynyboquinone (DNQ) kills a wide spectrum of cancer cell types(i.e., breast, non-small-cell lung, prostate, pancreatic) in anNQO1-dependent manner with greatly improved (20- to 100-fold) potencycompared to β-lapachone. DNQ lethality relies on NQO1-dependent futileredox cycling, using oxygen and generating extensive reactive oxygenspecies (ROS), particularly superoxide and hydrogen peroxide. ElevatedROS levels cause extensive DMA lesions and PAR-1 hyperactivation that,in turn, results in severe NAD′/ATP depletion that stimulatescalcium-dependent programmed necrotic cell death responses unique tothis class of NQO1 ‘bioactivated’ drugs (i.e., β-lapachone and DNQ).

A 2 hour exposure of NQO1+ cells to DNQ (LD₉₀: 50-250 nM) was sufficientfor complete cell death, while genetically match NQO1− cells wereunaffected. NQO1 or PARP-1 knockdown spared the short-term lethality ofDNQ-treated NQO1+ cells. BAPTA-AM (cytosolic Ca²⁺ chelator) and catalase(enzymatic H₂O₂ scavenger) rescued DNQ-induced lethality by long-termsurvival assessments. Thus, DNQ is a potent chemotherapeutic agentexhibiting a wide therapeutic window that holds great promise fortargeted therapy against a wide spectrum of difficult to treat cancers,including pancreatic and non-small cell lung cancer.

Despite considerable advances in cancer chemotherapy, the lack ofselectivity of most cancer chemotherapeutics remains a major limitingfactor. This disclosure describes the evaluation of elevatedNAD(P)H:quinone oxidoreductase-1 (NQO1, DT-diaphorase, EC 1.6.99.2)levels found in most solid tumors, particularly in non-small-cell lungcancer cells (NSCLC), prostate, pancreatic and breast, for developmentof therapeutic treatments. NQO1 is an inducible Phase II detoxifyingtwo-electron oxidoreductase capable of reducing most quinones, formingstable hydroquinones. In most cases, glutathione transferase thendetoxifies hydroquinones, conjugating them with glutathione forsecretion, and effectively avoiding more toxic seniquinones.

For some rare compounds, however, NQO1-mediated bioreduction can beexploited for antitumor activity. Rather than promoting detoxification,NQO1 activity can convert specific quinones into highly cytotoxicspecies. Most antitumor quinones dependent on NQO1 are DNA alkylators:(a) mitomycin C (MMC); (b) RH1; (c) E09; and (d) AZQ. However, these DNAalkylators are not only subject to detoxification pathways, butresistance from elevated or inducible DNA repair pathways limit theirusefulness. Furthermore, many of these drugs are efficient substratesfor one-electron oxidoreductases ubiquitously expressed in normaltissues.

The ortho-naphthoquinone, β-lapachone (β-lap, Scheme 1), kills culturedcancer cells and murine xenograft and orthotopic human or mouse tumormodels in vivo in an NQO1-dependent mariner. In contrast to alkylatingquinones, β-lap induces cell death by NQO1-dependent reactive oxygenspecies (ROS) formation and oxidative stress. NQO1 metabolism of β-lapresults into an unstable hydroquinone that is spontaneously oxidized bytwo equivalents of dioxygen, generating superoxide.

A futile cycle of oxidoreduction is thus established, and elevatedsuperoxide levels, in turn cause massive DNA base and single strandbreak (SSBs) lesions that normally are easily and rapidly repaired.However, extensive DNA lesions created in β-lap-treated NQO1over-expressing cancer cells results in hyperactivation ofpoly(ADP-ribose)polymerase-1 (PARP1), an otherwise essential base andSSB repair enzyme. In turn, PARP1 hyperactivation results in dramaticreduction of the NAD⁺/ATP pool due to ADP-ribosylation, causingtremendous energy depletion and cell death. As a result, β-lap killsNQO1+ cancer cells by a unique programmed necrosis mechanism that is:(a) independent of caspase activation or p53 status; (b) independent ofbcl-2 levels; (c) not affected by BAX/BAK deficiencies; (d) independentof EGFR, Ras or other constitutive signal transduction activation;and/or (e) not dependent on proliferation, since NQO1 is expressed inall cell cycle phases. Thus, β-lap is an attractive experimentalchemotherapeutic, and various β-lap formulations have been, or are in,phase I/II clinical trials.

However, β-lap has a fairly low potency (LD₅₀: 2-10 μM) in vitro, andhas limited aqueous solubility that complicates formulation anddelivery. Although nanoparticle strategies for β-lap delivery solvedformulations issues, resulting in promising antitumor efficacy, there isa clear need for better compounds to efficiently exploit NQO1over-expression in solid tumors.

Deoxynyboquinone (DNQ, Scheme 1) is a promising anti-neoplastic agentwhose mechanism of action has not been elucidated. Prior data indicatedthat DNQ killed cancer cells through oxidative stress and ROS formation.The cytotoxicity of DNQ was partially prevented by N-acetylcysteine, aglobal free radical scavenger and precursor to glutathione. It has nowbeen show that DNQ undergoes an NQO1-dependent futile cycle similar toβ-lap, where oxygen is consumed, ROS is formed and extensive DNA damagetriggers PARP1 hyperactivation, with dramatic decreases in essentialNAD⁺/ATP nucleotide pools, indicative of programmed necrosis.Importantly, DNQ is 20- to 100-fold more potent than β-lap, with asignificantly enhanced therapeutic window in NQO1+ versus NQO1− NSCLCcells. Efficacious NQO1-dependent killing by DNQ is also shown inbreast, prostate, and pancreatic cancer models in vitro. Furthermore, weshow that in vitro NQO1 processes DNQ much more efficiently than β-lap,suggesting that increased utilization accounts for its increasedpotency. Thus, DNQ offers significant promise as a selectivechemotherapeutic agent for the treatment of solid tumors with elevatedNQO1 levels.

Because NQO1 is overexpressed in the majority of solid tumors, and thecytotoxicity of DNQ depends predominately on the elevated expression ofthe enzyme NQO1, DNQ and its derivatives can be an excellent way toapproach targeting solid tumors. The invention provides numerous newcytotoxic compounds that can be used as new cancer therapeutics.

The foregoing and other objects and features of the disclosure willbecome more apparent from the following detailed description, whichproceeds with reference to the accompanying figures. Furtherembodiments, forms, features, aspects, benefits, objects, and advantagesof the present application shall become apparent from the detaileddescription and figures provided herewith.

Definitions

In general, the terms and phrases used herein have their art-recognizedmeaning, which can be found by reference to standard texts, journalreferences and contexts known to those skilled in the art. Suchart-recognized meanings may be obtained by reference to technicaldictionaries, such as Hawley's Condensed Chemical Dictionary 14^(th)Edition, by R. J. Lewis, John Wiley & Sons, New York, N.Y., 2001.

The singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, a referenceto “a compound” includes a plurality of such compounds, so that acompound X includes a plurality of compounds X. It is further noted thatthe claims may be drafted to exclude any optional element. As such, thisstatement is intended to serve as antecedent basis for the use ofexclusive terminology, such as “solely/” “only,” and the like, inconnection with the recitation of claim elements or use of a “negative”limitation.

The term “and/or” means any one of the items, any combination of theitems, or all of the items with which this term is associated. Thephrase “one or more” is readily understood by one of skill in the art,particularly when read in context of its usage. For example, one or moresubstituents on a phenyl ring refers to one to five, or one to four, forexample if the phenyl ring is disubstituted.

The term “about” can refer to a variation of ±5%, ±10%, ±20%, or ±25% ofthe value specified. For example, “about 50” percent can in someembodiments carry a variation from 45 to 55 percent. For integer ranges,the term “about” can include one or two integers greater than and/orless than a recited integer at each end of the range. Unless indicatedotherwise herein, the term “about” is intended to include values, e.g.,weight percents, proximate to the recited range that are equivalent interms of the functionality of the individual ingredient, thecomposition, or the embodiment.

As will be understood by the skilled artisan, all numbers, includingthose expressing quantities of ingredients, properties such as molecularweight, reaction conditions, and so forth, are approximations and areunderstood as being optionally modified in all instances by the term“about.” These values can vary depending upon the desired propertiessought to be obtained by those skilled in the art utilizing theteachings of the descriptions herein. It is also understood that suchvalues inherently contain variability necessarily resulting from thestandard deviations found in their respective testing measurements.

While the present invention can take many different forms, for thepurpose of promoting an understanding of the principles of theinvention, reference will now be made to the embodiments illustrated inthe drawings and specific language will be used to describe the same. Itwill nevertheless be understood that no limitation of the scope of theinvention is thereby intended. Any alterations and further modificationsof the described embodiments and any further applications of theprinciples of the invention as described herein are contemplated aswould normally occur to one skilled in the art to which the inventionrelates.

When a group of substituents is disclosed herein, it is understood thatall individual members of that group and all subgroups, including anyisomers, enantiomers, and diastereomers of the group members, aredisclosed separately. When a Markush group or other grouping is usedherein, all individual members of the group and all combinations andsubcombinations possible of the group are intended to be individuallyincluded in the disclosure. When a compound is described herein suchthat a particular isomer, enantiomer or diastereomer of the compound isnot specified, for example, in a formula or in a chemical name, thatdescription is intended to include each isomers and enantiomer of thecompound described individual or in any combination. Additionally,unless otherwise specified, all isotopic variants of compounds disclosedherein are intended to be encompassed by the disclosure. For example, itwill be understood that any one or more hydrogens in a moleculedisclosed can be replaced with deuterium or tritium. Isotonic variantsof a molecule are generally useful as standards in assays for themolecule and in chemical and biological research related to the moleculeor its use. Methods for making such isotopic variants are known in theart. Specific names of compounds are intended to be exemplary, as it isknown that one of ordinary skill in the art can name the same compoundsdifferently.

Whenever a range is given in the specification, for example, atemperature range, a time range, a carbon chain range, or a compositionor concentration range, all intermediate ranges and subranges, as wellas all individual values included in the ranges given are intended to beindividually included in the disclosure. It will be understood that anysubranges or individual values in a range or subrange that are includedin the description can be optionally excluded from embodiments of theinvention.

As used herein, “comprising” is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps. As usedherein, “consisting of” excludes any element, step, or ingredient notspecified in the claim element. As used herein, “consisting essentiallyof” does not exclude materials or steps that do not materially affectthe basic and novel characteristics of the claim. In each instanceherein any of the terms “comprising”, “consisting essentially of” and“consisting of” may be replaced with either of the other two terms. Theinvention illustratively described herein suitably may be practiced inthe absence of any element or elements, limitation or limitations whichis not specifically disclosed herein.

A “chemotherapeutic agent” refers to any substance capable of reducingor preventing the growth, proliferation, or spread of a cancer cell, apopulation of cancer cells, tumor, or other malignant tissue. The termis intended also to encompass any antitumor or anticancer agent.

A “therapeutically effective amount” of a compound with respect to thesubject method of treatment refers to an amount of the compound(s) in apreparation which, when administered as part of a desired dosage regimen(to a mammal, such as a human) alleviates a symptom, ameliorates acondition, or slows the onset of disease conditions according toclinically acceptable standards for the disorder or condition to betreated or the cosmetic purpose, e.g., at a reasonable benefit/riskratio applicable to any medical treatment.

The terms “treating”, “treat” and “treatment” include (i) preventing adisease, pathologic or medical condition from occurring (e.g.,prophylaxis); (ii) inhibiting the disease, pathologic or medicalcondition or arresting its development; (iii) relieving the disease,pathologic or medical condition; and/or (iv) diminishing symptomsassociated with the disease, pathologic or medical condition. Thus, theterms “treat”, “treatment”, and “treating” can extend to prophylaxis andcan include prevent, prevention, preventing, lowering, stopping orreversing the progression or severity of the condition or symptoms beingtreated. As such, the terns “treatment” can include medical,therapeutic, and/or prophylactic administration, as appropriate. Theterns “treating” or “treatment” can include reversing, reducing, orarresting the symptoms, clinical signs, and underlying pathology of acondition in manner to improve or stabilize a subject's condition.

The terms “inhibit”, “inhibiting”, and “inhibition” refer to theslowing, halting, or reversing the growth or progression of a disease,infection, condition, or group of cells. The inhibition can be greaterthan about 20%, 40%, 60%, 80%, 90%, 95%, or 99%, for example, comparedto the growth or progression that occurs in the absence of the treatmentor contacting.

The term “contacting” refers to the act of touching, making contact, orof bringing to immediate or close proximity. Including at the cellularor molecular level, for example, to bring about a physiologicalreaction, a chemical reaction, or a physical change, e.g., in asolution. In a reaction mixture, in vitro, or in vivo.

The term “exposing” is intended to encompass definitions as broadlyunderstood in the art. In an embodiment, the term means to subject orallow to be subjected to an action, influence, or condition. For exampleand by way of example only, a cell can be subjected to the action,influence, or condition of a therapeutically effective amount of apharmaceutically acceptable form of a chemotherapeutic agent.

The term “cancer cell” is intended to encompass definitions as broadlyunderstood in the art. In an embodiment, the term refers to anabnormally regulated cell that can contribute to a clinical condition ofcancer in a human or animal. In an embodiment, the term can refer to acultured cell line or a cell within or derived from a human or animalbody. A cancer cell can be of a wide variety of differentiated cell,tissue, or organ types as is understood in the art.

The term “tumor” refers to a neoplasm, typically a mass that includes aplurality of aggregated malignant cells.

The following groups can be R groups or bridging groups, as appropriate.

The term “alkyl” refers to a monoradical branched or unbranchedsaturated hydrocarbon chain preferably having from 1 to 30 carbon atoms.Short alkyl groups are those having 1 to 12 carbon atoms includingmethyl, ethyl, propyl, butyl, pentyl and hexyl groups, including allisomers thereof. Long alkyl groups are those having 12-30 carbon atoms.The group may be a terminal group or a bridging group.

Alkyl, heteroalkyl, aryl, heteroaryl, and heterocycle groups, and cyclicand/or unsaturated versions thereof, can be R groups of Formula I, andeach group can be optionally substituted.

The term “substituted” indicates that one or more hydrogen atoms on thegroup indicated in the expression using “substituted” is replaced with a“substituent”. The number referred to by ‘one or more’ can be apparentfrom the moiety one which the substituents reside. For example, one ormore can refer to, e.g., 1, 2, 3, 4, 5, or 6; in some embodiments 1, 2,or 3; and in other embodiments 1 or 2. The substituent can be one of aselection of indicated groups, or it can be a suitable group known tothose of skill in the art, provided that the substituted atom's normalvalency is not exceeded, and that the substitution results in a stablecompound. Suitable subsequent groups include, e.g., alkyl, alkenyl,alkynyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, aroyl,(aryl)alkyl (e.g., benzyl or phenylethyl), heteroaryl, heterocycle,cycloalkyl, alkanoyl, alkoxycarbonyl, amino, alkylamino, dialkylamino,trifluoromethyl, trifluoromethoxy, trifluoromethylthio, difluoromethyl,acylamino, nitro, carboxy, carboxyalkyl, keto, thioxo, alkylthio,alkylsulfinyl, alkylsulfonyl, arylsulfinyl, arylsulfonyl,heteroarylsulfinyl, heteroarylsulfonyl, heterocyclesulfinyl,heterocyclesulfonyl, phosphate, sulfate, hydroxyl amine, hydroxyl(alkyl)amine, and cyano. Additionally, suitable substituent groups canbe, e.g., —X, —R, —O⁻, —OR, —SR, —S⁻, —NR₂, —NR₃, ═NR, —CX₃, —CN, —OCN,—SCN, —N═C═O, —NCS, —NO, —NO₂, ═N₂, —N₃, —NC(═O)R, —C(═O)R, —C(═O)NRR,—S(═O)₂O⁻, —S(═O)₂OH, —S(═O)₂R, —OS(═O)₂OR, —S(═O)₂NR, —S(═O)R,—OP(═O)O₂RR, —P(═O)O₂RR, —P(═O)(O⁻)₂, —P(═O)(OH)₂, —C(═O)R, —C(═O)X,—C(S)R, —C(O)OR, —C(O)O⁻, —C(S)OR, —C(O)SR, —C(S)SR, —C(O)NRR, —C(S)NRR,or —(NR)NRR, where each X is independently a halogen (“halo”): F, Cl,Br, or I; and each R is independently H, alkyl, aryl, (aryl)alkyl (e.g.,benzyl), heteroaryl, (heteroaryl)alkyl, heterocycle, heterocycle(alkyl),or a protecting group. As would be readily understood by one skilled inthe art, when a substituent is keto (═O) or thioxo (═S), or the like,then two hydrogen atoms on the substituted atom are replaced. In someembodiments, one or more of the substituents above can be excluded fromthe group of potential values for substituents on a substituted group.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, often having from 2to 14 carbons, or 2 to 10 carbons in the chain, including at least onecarbon atom and at least one heteroatom selected from the groupconsisting of O, N, P, Si and S, and wherein the nitrogen and sulfuratoms may optionally be oxidized and the nitrogen heteroatom mayoptionally be quaternized. The heteroatom(s) O, N, P and S and Si may beplaced at any interior position of the heteroalkyl group or at theposition at which the alkyl group is attached to the remainder of themolecule. The heteroalkyl group can have, for example, one to about 20carbon atoms in a chain. Examples include, but are not limited to,—CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃,—CH₂—CH₂—S(O)—CH₃, —CH₂CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃,—CH₂—CH═N—OCH₃, —CH═CH—N(CH₃)—CH₃, O—CH₃, —O—CH₂—CH₃, and —CH.Additional examples of heteroalkyl groups include alkyl ethers,secondary and tertiary alkyl amines, amides, alkyl sulfides, and thelike. The group may be a terminal group or a bridging group. As usedherein, reference to a chain when used in the context of a bridginggroup refers to the direct chain of atoms linking the two terminalpositions of the bridging group.

The term “alcohol” as used herein may be defined as an alcohol thatcomprises a C₁₋₁₂ alkyl moiety substituted at a hydrogen atom with onehydroxyl group. Alcohols include ethanol, n-propanol, i-propanol,n-butanol, i-butanol, s-butanol, t-butanol, n-pentanol, i-pentanol,n-hexanol, cyclohexanol, n-heptanol, n-octanol, n-nonanol, n-decanol,and the like. The carbon atoms in alcohols can be straight, branched orcyclic.

“Acyl” may be defined as an alkyl-CO-group in which the alkyl group isas described herein. Examples of acyl include acetyl and benzoyl. Thealkyl group can be a C₁-C₆ group. The group may be a terminal group or abridging (i.e., divalent) group.

“Alkoxy” refers to an —O-alkyl group in which alkyl is defined herein.Preferably the alkoxy is a C₁-C₆alkoxy. Examples include, but are notlimited to, methoxy and ethoxy. The group may be a terminal group or abridging group.

“Alkenyl” as a group or part of a group denotes an aliphatic hydrocarbongroup containing at least one carbon-carbon double bond and which may bestraight or branched preferably having 2-14 carbon atoms, morepreferably 2-12 carbon atoms, most preferably 2-6 carbon atoms, in thenormal chain. The group may contain a plurality of double bonds in thenormal chain and the orientation about each is independently E or Z.Exemplary alkenyl groups include, but are not limited to, ethenyl,propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl and nonenyl. Thegroup may be a terminal group or a bridging group.

“Alkynyl” as a group or part of a group may be defined as an aliphatichydrocarbon group containing a carbon-carbon triple bond, the chair ofwhich may be straight or branched preferably having from 2-14 carbonatoms, more preferably 2-12 carbon atoms, more preferably 2-6 carbonatoms in the normal chain. Exemplary structures include, but are notlimited to, ethynyl and propynyl. The group may be a terminal group or abridging group,

“Alkenyloxy” refers to an —O-alkenyl group in which alkenyl is asdefined herein. Preferred alkenyloxy groups are C₁-C₆ alkenyloxy groups.The group may be a terminal group or a bridging group.

“Alkynyloxy” refers to an —O-alkynyl group in which alkynyl is asdefined herein. Preferred alkynyloxy groups are C₁-C₆ alkynyloxy groups.The group may be a terminal group or a bridging group.

“Alkoxycarbonyl” refers to an —C(O)—O-alkyl group in which alkyl is asdefined herein. The alkyl group is preferably a C₁-C₆ alkyl group.Examples include, but not limited to, methoxycarbonyl andethoxycarbonyl. The group may be a terminal group or a bridging group.

“Alkylsulfinyl” may be defined as a —S(O)-alkyl group in which alkyl isas defined above. The alkyl group is preferably a C₁-C₆ alkyl group.Exemplary alkylsulfinyl groups include, but not limited to,methylsulfinyl and ethylsulfinyl. The group may be a terminal group or abridging group.

“Alkylsulfonyl” refers to a —S(O)₂-alkyl group in which alkyl is asdefined above. The alkyl group is preferably a C₁-C₆ alkyl group.Examples include, but not limited to methylsulfonyl and ethylsulfonyl.The group may be a terminal group or a bridging group.

“Amino” refers to —NH₂, and “alkylamino” refers to —NR₂, wherein atleast one R is alkyl and the second R is alkyl or hydrogen. The term“acylamino” refers to RC(═O)NH—, wherein R is alkyl or aryl. The alkylgroup can be, for example, a C₁-C₆ alkyl group. Examples include, butare not limited to methylamine and ethylamino. The group may be aterminal group or a bridging group.

“Alkylaminocarbonyl” refers to an alkylamino-carbonyl group in whichalkylamino is as defined above. The group may be a terminal group or abridging group.

“Cycloalkyl” refers to a saturated or partially saturated, monocyclic orfused or spiro polycyclic, carbocycle of 3 to about 30 carbon atoms,often containing 3 to about 9 carbons per ring, such as cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, and the like. Itincludes monocyclic systems such as cyclopropyl and cyclohexyl, bicyclicsystems such as decalin, and polycyclic systems such as adamantane. Thegroup may be a terminal group or a bridging group.

“Cycloalkenyl” may be defined as a non-aromatic monocyclic ormulticyclic ring system containing at least one carbon-carbon doublebond and preferably having from 5-40 carbon atoms per ring. Exemplarymonocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl orcycloheptenyl. The cycloalkenyl group may be substituted by one or moresubstituent groups. The group may be a terminal group or a bridginggroup.

Alkyl and cycloalkyl groups can be substituents on the alkyl portions ofother groups, such as without limitation, alkoxy, alkyl amines, alkylketones, arylalkyl, heteroarylalkyl, alkylsulfonyl and alkyl estersubstituents and the like. The group may be a terminal group or abridging group.

“Cycloalkylalkyl” may be defined as a cycloalkyl-alkyl-group in whichthe cycloalkyl and alkyl moieties are as previously described. Exemplarymonocycloalkylalkyl groups include cyclopropylmethyl, cyclopentylmethyl,cyclohexylmethyl and cycloheptylmethyl. The group may be a terminalgroup or a bridging group.

“Heterocycloalkyl” refers to a saturated or partially saturatedmonocyclic, bicyclic, or polycyclic ring containing at least oneheteroatom selected from nitrogen, sulfur, oxygen, preferably from 1 to3 heteroatoms in at least one ring. Each ring is preferably from 3 to 10membered, more preferably 4 to 7 membered. Examples of suitableheterocycloalkyl substituents include pyrrolidyl, tetrahydrofuryl,tetrahydrothiofuranyl, piperidyl, piperazyl, tetrahydropyranyl,morpholino, 1,3-diazapane, 1,4-diazapane, 1,4-oxazepane, and1,4-oxathiapane. The group may be a terminal group or a bridging group.

“Heterocycloalkenyl” refers to a heterocycloalkyl as described above butcontaining at least one double bond. The group may be a terminal groupor a bridging group.

“Heterocycloalkylalkyl” refers to a heterocycloalkyl-alkyl group inwhich the heterocycloalkyl and alkyl moieties are as previouslydescribed. Exemplary heterocycloalkylalkyl groups include(2-tetrahydrofuryl)methyl, and (2-tetrahydrothiofuranyl)methyl. Thegroup may be a terminal group or a bridging group.

“Halo” refers to a halogen substituent such as fluoro, chloro, bromo, oriodo.

The term “aryl” refers to an aromatic hydrocarbon group derived from theremoval of one hydrogen atom from a single carbon atom of a parentaromatic ring system. The radical can be at a saturated or unsaturatedcarbon atom of the parent ring system. The aryl group can have from 6 to18 carbon atoms. The aryl group can have a single ring (e.g., phenyl) ormultiple condensed (fused) rings, wherein at least one ring is aromatic(e.g., naphthyl, dihydrophenanthrenyl, fluorenyl, or anthryl). Typicalaryl groups include, but are nor limited to, radicals derived frombenzene, naphthalene, anthracene, biphenyl, and the like. The aryl canbe unsubstituted or optionally substituted, as described above for alkylgroups.

The term “heteroaryl” is defined herein as a monocyclic, bicyclic, ortricyclic ring system containing one, two, or three aromatic rings andcontaining at least one nitrogen, oxygen, or sulfur atom in an aromaticring, and which can be unsubstituted or substituted, for example, withone or more, and in particular one to three, substituents, as describedabove in the definition of “substituted”. Examples of heteroaryl groupsinclude, but are not limited to, 2H-pyrrolyl, 3H-indolyl,4H-quinolizinyl, acridinyl, benzo[b]thienyl, benzothiazolyl,β-carbolinyl, carbazolyl, chromenyl, cinnolinyl, dibenzo[b,d]furanyl,furazanyl, furyl, imidazolyl, imidizolyl, indazolyl, indolisinyl,indolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl,isoxazolyl, naphthyridinyl, oxazolyl, perimidinyl, phenanthridinyl,phenanthrolinyl, phenarsazinyl, phenazinyl, phenothiazinyl,phenoxathlinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl,pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl,pyrimidinyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl,thiadiazolyl, thianthrenyl, thiazolyl, thienyl, triazolyl, tetrazolyl,and xanthenyl. In one embodiment the terms “heteroaryl” denotes amonocyclic aromatic ring containing five or six ring atoms containingcarbon and 1, 2, 3, or 4 heteroatoms independently selected fromnon-peroxide oxygen, sulfur, and N(Z) wherein Z is absent or is H, O,alkyl, aryl, or (C₁-C₆)alkylaryl. In another embodiment heteroaryldenotes an ortho-fused bicyclic heterocycle of about eight to ten ringatoms derived therefrom, particularly a benz-derivative or one derivedby fusing a propylene, trimethylene, or tetramethylene diradicalthereto.

The term “heterocycle” refers to a saturated or partially unsaturatedring system, containing at least one heteroatom selected from the groupoxygen, nitrogen, and sulfur, and optionally substituted with one ormore groups as defined herein under the term “substituted”. Aheterocycle can be a monocyclic, bicyclic, or tricyclic group containingone or more heteroatoms. A heterocycle group also can contain an oxogroup (═O) attached to the ring. Non-limiting examples of heterocyclegroups include 1,3-dihydrobenzofuran, 1,3-dioxolane, 1,4-dioxane,1,4-dithiane, 2H-pyran, 2-pyrazoline, 4H-pyran, chromanyl,imidazolidinyl, imidazolinyl, indolinyl, isochromanyl, isoindolinyl,morpholine, piperazinyl, piperidine, piperidyl, pyrazolidine,pyrazolidinyl, pyrazolinyl, pyrrolidine, pyrroline, quinuclidine, andthiomorpholine.

The abbreviation “DNQ_(d)” as used herein refers to an analog orderivative of DNQ.

Additional groups that can be bridging groups or terminal groups of R₁,R₂, R₃ and R₄ are described below.

The term “carbonate ester” may be defined as a functional group having ageneral structure R′CO(═O)OR, where R′ can be the tricyclic core ofFormula I and R can be as defined in the definitions of the variables ofFormula I.

The term “ester” may be defined as a functional group having a generalstructure RC(═O)OR′, where R′ can be the tricyclic core of Formula I andR can be as defined in the definitions of the variables of Formula I, orvice versa.

The term “hemiacetal” may be defined as a functional group having ageneral structure

where R′ can be the tricyclic core of Formula I and R can be as definedin the definitions of the variables of Formula I, or vice versa.

The term “carboxamide” may be defined as a functional group having ageneral structure

where R′ can be the tricyclic core of Formula I and R can be as definedin the definitions of the variables of Formula I, or vice versa, and R″can be R can be as defined in the definitions of the variables ofFormula I.

The term “imine” may be defined as a functional group having generalstructures

where R′ can be the tricyclic core of Formula I and R can be as definedin the definitions of the variables of Formula I, or vice versa, and R″can be the tricyclic core of Formula I or R can be as defined in thedefinitions of the variables of Formula I.

A “pyridyl” group can be a 2-pyridyl, 3-pyridyl, or 4-pyridyl group.

The term “imide” may be defined as a functional group having a generalstructure

where R′ can be the tricyclic core of Formula I and R can be as definedin the definitions of the variables of Formula I, or vice versa, and R″can be the tricyclic core of Formula I or R can be as defined in thedefinitions of the variables of Formula I.

The term “sulhydryl” may be defined as a functional group having ageneral structure —S—H.

The term “sulfinyl” may be defined as a functional group having ageneral structure

where R′ can be the tricyclic core of formula I and R can be as definedin the definitions of the variables of Formula I, or vice versa.

The term “sulfonyl” may be defined as a functional group having ageneral structure

where R′ can be the tricyclic core of Formula I and R can be as definedin the definitions of the variables of Formula I, or vice versa.

The term “phosphate” may be defined as a functional group having generalstructures

The term “phosphono” may be defined as a functional group having ageneral structure

The term “hexose” may be defined as a monosaccharide having six carbonatoms having the general chemical formula C₆H₁₂O₆ and can includealdohexoses which have an aldehyde functional group at position 1 orketohexoses which have a ketone functional group at position 2. Examplealdohexoses include, allose, altrose, glucose, mannose, gulose, idose,galactose, and talose, in either D or L form.

Compounds and Methods of the Invention

The invention provides DNQ compounds and methods of using suchcompounds. Accordingly, the invention provides compounds of Formula (I):

wherein

R₁, R₂, R₃, and R₄ are each independently —H or —X—R;

each X is independently a direct bond or a bridging group, wherein thebridging group is —O—, —S—, —NH—, —C(═O)—, —O—C(═O)—, —C(═)—O—,—O—C(═O)—O—, or a linker of the formula -W-A-W-, wherein

each W is independently —N(R′)C(═O)—, —C(═O)N(R′)—, —OC(═O)—, —C(═O)O—,—O—, —S—, —S(O)—, —S(O)₂—, —N(R′)—, —C(═O)—, —(CH₂)_(n)— where n is1-10, or a direct bond, wherein each R′ is independently H,(C₁-C₆)alkyl, or a nitrogen protecting group; and

each A is independently (C₁-C₂₀)alkyl, (C₂-C₁₆)alkenyl, (C₂-C₁₆)alkynyl,(C₃-C₈)cycloalkyl, (C₆-C₁₀)aryl, —(OCH₂—CH₂)_(n)— where n is 1 to about20, —C(O)NH(CH₂)_(n)— wherein n is 1 to about 6, —OP(O)(OH)O—,—OP(O)(OH)O(CH₂)_(n)— wherein n is 1 to about 6, or (C₁-C₂₀)alkyl,(C₂-C₁₆)alkenyl, (C₂-C₂₆)alkynyl, or —(OCH₂—CH₂)_(n)— interruptedbetween two carbons, or between a carbon and an oxygen, with acycloalkyl, heterocycle, or aryl group;

each R is independently alkyl, alkenyl, alkynyl, heteroalkyl,cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl,(cycloalkyl)alkyl, (heterocycloalkyl)alkyl, (cycloalkyl)heteroalkyl,(heterocycloalkyl)heteroalkyl, aryl, heteroaryl, (aryl)alkyl,(heteroaryl)alkyl, hydrogen, hydroxy, hydroxyalkyl, alkoxy,(alkoxy)alkyl, alkenyloxy, alkynyloxy, (cycloalkyl)alkoxy,heterocycloalkyloxy, amino, alkylamino, aminoalkyl, acylamino,arylamino, sulfonylamino, sulfinylamino, —COR^(x), —COOR^(x),—CONHR^(x), —NHCOR^(x), —NHCOOR^(x), —NHCONHR^(x), —N₃, —CN, —NC, —NCO,—NO₂, —SH, -halo, alkoxycarbonyl, alkylaminocarbonyl, sulfonate,sulfonic acid, alkylsulfonyl, alkylsulfinyl, arylsulfonyl, arylsulfinyl,aminosulfonyl, R^(x)S(O)R^(y)—, R^(x)S(O)₂R^(y)—,R^(x)C(O)N(R^(x))R^(y)—, R^(x)SO₂N(R^(x))R^(y)—,R^(x)N(R^(x))C(O)R^(y)—, R^(x)N(R^(x))SO₂R^(y)—,R^(x)N(R^(x))C(O)N(R^(x))R^(y)—, carboxaldehyde, acyl, acyloxy, —OPO₃H₂,—OPO₃Z₂, where Z is an inorganic cation, or saccharide; where each R^(x)is independently H, OH, alkyl or aryl, and each R^(y) is independently agroup W;

wherein any alkyl or aryl can be optionally substituted with one or morehydroxy, amino, cyano, nitro, or halo groups:

or a salt or solvate thereof.

In some embodiments, when R₁, R₂, and R₃ are methyl, R₄ is not H ormethyl. In other embodiments, when R₁, R₃, and R₄ are methyl, the group—X—R of R₂ is not —CH₂—OAc. In certain embodiments, when R₁, R₃, and R₄are methyl, the R group of R₂ is not acyloxy. In various embodiments,R₁-R₄ are not each H. In certain embodiments, R₁-R₄ are not each alkyl,such as unsubstituted alkyl. In some embodiments, R₁-R₄ are not eachmethyl.

In one embodiment, R₁, R₂, R₃, and R₄ are each (C₁₋₂₀)alkyl groups. Insome embodiments, the (C₁₋₂₀)alkyl group is a (C₂₋₂₀)alkyl group, a(C₃₋₂₀)alkyl group, a (C₄₋₂₀)alkyl group, a (C₅₋₂₀)alkyl group, or a(C₁₀₋₂₀)alkyl group. The alkyl groups can be substituted, for example,with a hydroxyl or phosphate group. The phosphate group can be aphosphonic acid or a phosphonic acid salt, such as a lithium salt, asodium salt, a potassium salt, or other known salt of phosphonic acids.

A specific value for R₁ is H. A specific value for R₂ is H. A specificvalue for R₃ is H. A specific value for R₄ is H.

A specific value for R₁ is methyl. A specific value for R₂ is methyl. Aspecific value for R₃ is methyl. A specific value for R₄ is methyl. Themethyl can be substituted as described above for the term “substituted”.

In some embodiments of Formula (I):

-   R₁ and R₂ are methyl; R₃ is hydrogen; and R₄ is 2-methyl-propane;-   R₁ and R₂ are methyl; R₃ is hydrogen; and R₄ is butyl;-   R₁ and R₂ are methyl and R₃ is hydrogen; and R₂ is ethyl;-   R₁ and R₂ are methyl and R₃ is hydrogen; and R₄ is ethyl;-   R₁ is methyl; R₃ is hydrogen; R₂ is propyl; and R₄ is butyl;-   R₁ and R₄ are methyl; R₂ is propyl and R₃ is hydrogen;-   R₁ is propyl; R₂ and R₄ are methyl and R₃ is hydrogen;-   R₁ and R₂ are ethyl; R₃ is hydrogen; and R₂ is methyl;-   R₁ is propyl; R₂ is methyl; R₃ is hydrogen; and R₄ is butyl;-   R₁ and R₂ are propyl; R₃ is hydrogen; and R₄ is butyl;-   R₁ and R₂ are methyl; R₃ is hydrogen; and R₄ is C₁₂alkyl;-   R₁ and R₂ are methyl; R₃ is hydrogen; and R₄ is tert-butyl;-   R₁ and R₂ are methyl; R₃ is hydrogen; and R₄ is hydroxypropyl;-   R₁ and R₂ are methyl; R₃ is hydrogen; and R₄ is 3,3-dimethylbutyl    [—CH₂CH₂C(CH₃)₂CH₃];-   R₁ and R₂ are methyl; R₃ is hydrogen; and R₄ is 3-methybutyl    [—CH₂CH₂CH(CH₃)CH₃)];-   R₂ and R₄ are methyl; R₃ is hydrogen; and R₁ is ethyl;-   R₁ and R₂ are methyl; R₃ is hydrogen; and R₄ is propyl;-   R₁ and R₂ are methyl; R₃ is hydrogen; and R₄ is n-pentyl;-   R₁ and R₂ are methyl; R₃ is hydrogen; and R₄ is n-hexyl;-   R₁ and R₂ are methyl; R₃ is hydrogen; and R₄ is isopropyl;-   R₁ and R₂ are methyl; R₃ is hydrogen; and R₄ is cyclooctyl;-   R₁ and R₂ are methyl; R₃ is hydrogen; and R₄ is cyclopropyl;-   R₁ and R₂ are methyl; R₃ is hydrogen; and R₄ is methylcyclopropyl;-   R₁ and R₂ are methyl; R₃ is hydrogen; and R₄ is ethylcyclopropyl;-   R₁ is C₁₂alkyl; R₂ and R₄ are methyl; and R₃ is hydrogen;-   R₁ and R₄ are methyl; R₃ is hydrogen; and R₂ is C₁₂alkyl;-   R₁, R₂ , and R₃ are methyl; and R₄ is —CH₂OPO₃Na₂;-   R₁ is —CH₂OPO₃Na₂; R₂ and R₃ are methyl; and R₄ is hydrogen;-   R₁ and R₃ are methyl; R₂ is —CH₂OPO₃Na₂; and R₄ is hydrogen;-   R₁ and R₂ are methyl; R₃ is —CH₂OPO₃Na₂; and R₄ is hydrogen;-   R₁ and R₂ are methyl; R₃ is —CH₂OPO₃Na₂; and R₄ is hydrogen;-   R₁, R₂, and R₃ are methyl; and R₄ is —CH₂OH;-   R₁ is —CH₂OH; R₂ and R₃ are methyl; and R₄ is hydrogen;-   R₁ and R₃ are methyl; R₂ is —CH₂OH; and R₄ is hydrogen;-   R₁ and R₂ are methyl; R₃ is —CH₂OH; and R₄ is hydrogen; or    R₁ and R₂ are methyl; R₃ is —CH₂CH₂OH; and R₄ is hydrogen.    Additional specific compounds and formulas of the invention are    illustrated in FIGS. 1 and 2.

In certain embodiments of Formula I, R¹ is (C₁₋₄) alkyl group. Incertain instances, R¹ is (C₁₋₃) alkyl group. In certain instances, R¹ is(C₁₋₂) alkyl group.

In certain embodiments of Formula I, R² is (C₁₋₄) alkyl group. Incertain instances, R² is (C₁₋₃) alkyl group. In certain instances, R² is(C₁₋₂) alkyl group.

In certain embodiments of Formula I, R³ is hydrogen.

In certain embodiments of Formula I, R⁴ is an optionally substituted(C₁₋₁₀) alkyl group, where the alkyl group is substituted with hydroxyl,halogen, amino, or thiol. In certain instances, R⁴ is (C₁₋₁₀) alkylgroup, (C₁₋₈) alkyl group, (C₁₋₆) alkyl group, or (C₁₋₄) alkyl group. Incertain instances, R⁴ is (C₂₋₆) alkyl group. In certain instances, R⁴ isa substituted (C₁₋₁₀) alkyl group, substituted (C₁₋₈) alkyl group,substituted (C₁₋₆) alkyl group, or substituted (C₁₋₄) alkyl group, wherethe alkyl group is substituted with hydroxyl, halogen, amino, or thiol,in certain instances, R⁴ is an alkyl group is substituted with hydroxyl.In certain instances, R⁴ is an alkyl group is substituted with halogen.In certain instances, R⁴ is an alkyl group is substituted with amino. Incertain instances, R₄ is an alkyl group is substituted with thiol.

In certain embodiments of Formula I, R¹ and R₂ are independently (C₁₋₄)alkyl groups; R³ is hydrogen; and R₄ is an optionally substituted(C₁₋₁₀) alkyl group, where the alkyl group is substituted with hydroxyl,halogen, amino, and thiol.

In certain embodiments of Formula I, R¹ and R² are independently (C₁₋₂)alkyl groups; R³ is hydrogen; and R⁴ is an optionally substituted(C₁₋₁₀) alkyl group, where the alkyl group is substituted with hydroxyl,halogen, amino, and thiol.

In certain embodiments of Formula I, R¹ and R² are independently (C₁₋₂)alkyl groups; R³ is hydrogen; and R⁴ is (C₁₋₁₀) alkyl group.

In certain embodiments of Formula I, R¹ and R² are independently (C₁₋₂)alkyl groups; R³ is hydrogen; and R⁴ is (C₁₋₈) alkyl group.

In certain embodiments of Formula I, R¹ and R₂ are independently (C₁₋₂)alkyl groups; R³ is hydrogen; and R⁴ is (C₁₋₆) alkyl group.

In certain embodiments of Formula I, R₁ and R₂ are independently (C₁₋₂)alkyl groups; R³ is hydrogen; and R⁴ is (C₁₋₄) alkyl group.

In certain embodiments of Formula I, R¹ and R² are independently (C₁₋₂)alkyl groups; R³ is hydrogen; and R⁴ is (C₂₋₆) alkyl group.

In certain embodiments of Formula I, R¹ and R₂ are independently (C₁₋₂)alkyl groups; R³ is hydrogen; and R⁴ is a substituted (C₁₋₆) alkylgroup, where the alkyl group is substituted with hydroxyl, halogen,amino, and thiol.

In certain embodiments of Formula I, R¹ and R² are independently (C₁₋₂)alkyl groups; R³ is hydrogen; and R⁴ is a substituted (C₁₋₄) alkylgroup, where the alkyl group is substituted with hydroxyl, halogen,amino, and thiol.

In certain embodiments, a compound of Formula I is Compound 87 or a saltor solvate thereof:

In certain embodiments, a compound of Formula I is Compound 9-253 or asalt or solvate thereof:

In certain embodiments, a compound of Formula I is Compound 9-251 or asalt or solvate thereof:

In certain embodiments, a compound of Formula I is Compound 10-41 or asalt or solvate thereof:

In certain embodiments, a compound of Formula I is Compound 109 or asalt or solvate thereof:

In certain embodiments, a compound of Formula I is Compound 107 or asalt or solvate thereof:

In certain embodiments, a compound of Formula I is Compound 9-281 or asalt or solvate thereof:

In certain embodiments, a compound of Formula I is Compound 9-249 or asalt or solvate thereof:

In certain embodiments, a compound of Formula I is Compound 9-255 or asalt or solvate thereof:

In certain embodiments, a compound of Formula I is Compound 9-257 or asalt or solvate thereof:

The invention also provides a pharmaceutical composition comprising acompound of Formula (I) and a pharmaceutically acceptable diluent,excipient, or carrier. The carrier can be water, for example, in thepresence of hydroxypropyl-β-cyclodextrin (HPβCD). The solubility of thecompound can be increase by about 100 times, about 200 times, about 500times, about 1000 times, about 2000 times, or about 3000 times, comparedto the compounds solubility in water without HPβCD.

The invention further provides DNQ compounds of formula (II):

wherein

R₁ and R₂ are each independently C₁₋₃₀alkyl;

R₃ is hydrogen or C₁₋₃₀alkyl;

R₄ is C₁₋₃₀alkyl or C₃₋₃₀cycloalkyl-C₁₋₃₀alkyl;

where the C₁₋₃₀alkyl groups can include, for example, methyl, ethyl,n-propyl, isopropyl, butyl, isobutyl, sec- and tert-butyl, pentyl,hexyl, heptyl, octyl, nonyl, and the like;

where each R₁, R₂, or R₄ alkyl can be optionally functionalized withfunctional group R^(a);

where R^(a) is either a bridging group or a terminal group selectedfrom:

 (1) Hydroxyl,  (2) Carbonyl,  (3) Aldehyde,  (4) Haloformyl,  (5)Carbonate ester,  (6) Carboxyl,  (7) Ester,  (8) Hydroperoxy,  (9)Peroxy, (10) Phenyl, (11) Alkenyl, (12) Benzyl, (13) Alkynyl, (14)Ether, (15) Hemiacetal, (16) Hemiketal, (17) Acetal, (18) Ketal, (19)Orthoester, (20) Orthocarbonate ester, (21) Carboxamide, (22) Amine,(23) Ketamine, (24) Imide, (25) Azide, (26) Azo, (27) Cyanate, (28)Isocyanate, (29) Nitrate, (30) Nitrile, (31) Isonitrile, (32)Nitrosooxy, (33) Nitro, (34) Nitroso, (35) Pyridyl, (36) Sulfhydryl,(37) Sulfide, (38) Disulfide, (39) Sulfinyl, (40) Sulfonyl, (41)Sulfino, (42) Sulfo, (43) Thiocyanate, (44) Isothiocyanate, (45)Carbonothioyl, (46) Phosphine, (47) Phosphono, (48) Phosphate, (49)Halogen; or (50) Hexose;or a pharmaceutical acceptable salt or solvate thereof.

In some embodiments, when R₁, R₂, and R₄ are methyl, R₃ is not hydrogenor methyl.

As to any of the above formulas or groups that contain one or moresubstituents, it is understood, of course, that such groups do notcontain any substitution or substitution patterns that are stericallyimpractical and/or synthetically non-feasible. In addition, thecompounds of this invention include all stereochemical isomers arisingfrom the substitution of these compounds.

Selected substituents of the compounds described herein may be presentto a recursive degree. In this context, “recursive substituent” meansthat a substituent may recite another instance of itself. Because of therecursive nature of such substituents, theoretically, a large number maybe present in any given claim. One of ordinary skill in the art ofmedicinal chemistry and organic chemistry understands that the totalnumber of such substituents is reasonably limited by the desiredproperties of the compound intended. Such properties include, by ofexample and not limitation, physical properties such as molecularweight, solubility or log P, application properties such as activityagainst the intended target, and practical properties such as ease ofsynthesis.

Recursive substituents are an intended aspect of the invention. One ofordinary skill in the art of medicinal and organic chemistry understandsthe versatility of such substituents. To the degree that recursivesubstituents are present in a claim of the invention, the total numberwill be determined as set forth above. In some embodiments, recursivesubstituents are present only to the extent that the molecular mass ofthe compound is about 400 to about 1600, about 450 to about 1200, about500 to about 100, about 600 to about 800. In other embodiments,recursive substituents are present only to the extent that the molecularmass of the compound is less than 2000, less than 1800, less than 1600,less than 1500, less than 1400, less than 1200, less than 1000, lessthan 900, less than 800, less than 750, less than 700, or less thanabout 600.

Patients with solid tumors having elevated NQO1 levels can be treatedthrough the administration of an effective amount of a pharmaceuticallyactive form of DNQ and/or DNQ_(d) (DNQ compounds). DNQ and DNQ_(d)compounds can be, for example, a compound defined by one of the formulasof FIG. 1, or a compound illustrated in FIG. 2. In FIG. 1 where n=1-30,the value of n can be 1 or any integer from 1 up to about 30. Thus, therange 1-30 includes each individual integer from 1 to 30 and any rangesfrom any one to any second number from 1 to 30. In each range describedherein, a portion of the range may also be excluded from the embodimentdefined. For example, in various embodiments, a variable n can be 6-24,and another n variable of the same formula can be 1-24.

In FIG. 1 for DNQ_(d)-20, R₁, R₂, and R₃ can be as defined for Formula Iabove. In various embodiments, R₁, R₂, and R₃ can also eachindependently be C₁₋₂₀alkyl, or each of R₁, R₂ or R₃ can beindependently be linked to the anomeric position of a hexose, optionallythrough a linker, such as a linker of formula -W-A-W- or a(C₁-C₁₀)alkylene group.

In FIG. 1 for DNQ_(d)-27 and DNQ_(d)-28, X can be a linker of formula-W-A-W- or a divalent bridging group such as a divalent alkyl, alkenyl,alkynyl, heteroalkyl, acycloalkyl, cycloalkenyl, heterocycloalkyl,heterocycloalkenyl, cycloalkylalkyl, heterocycloalkylalkyl,cycloalkylheteroalkyl, heterocycloalkylheteroalkyl, alkoxy, alkoxyalkyl,alkenyloxy, alkynyloxy, cycloalkylkoxy, heterocycloalkyloxy, amino,alkylamino, aminoalkyl, acylamino, arylamino, sulfonylamino,sulfinylamino, alkoxycarbonyl, alkylaminocarbonyl, sulfonyl,alkylsulfonyl, alkylsulfinyl, arylsulfonyl, arylsulfinyl, aminosulfonyl,or acyl, each of which may be optionally substituted.

In FIG. 1 for DNQ_(d)-29, each X can independently be a linker offormula -W-A-W- or a divalent bridging group as described above forDNQ_(d)-27 and DNQ_(d)-28; and each Y can independently be:

 (1) Hydroxyl,  (2) Aldehyde,  (3) Carboxyl,  (4) Haloformyl,  (5)Hydroperoxy,  (6) Phenyl,  (7) Benzyl,  (8) Alkyl,  (9) Alkenyl, (10)Alkynyl, (11) Acetate, (12) Amino, (13) Azide, (14) Azo, (15) Cyano,(16) Isocyanato, (17) Nitrate, (18) Isonitrile, (19) Nitrosooxy, (20)Nitro, (21) Nitroso, (22) Pyridyl, (23) Sulfhydryl, (24) Sulfonic acid,(25) Sulfonate, (26) Isothiocyanato, (27) Phosphine, (28) Phosphate,(29) Halo, or (30) Hexose.

The invention also provides methods of treating a patient that has tumorcells having elevated NQO1 levels. The methods can include administeringto a patient having tumor cells with elevated NQO1 levels atherapeutically effective amount of a compound of Formula (I), or acomposition described herein. The invention further provides methods oftreating a tumor cell having an elevated NQO1 level comprising exposingthe tumor cell to a therapeutically effective amount of a compound orcomposition described herein, wherein the tumor cell is treated, killed,or inhibited from growing. The tumor or tumor cells can be malignanttumor cells, in some embodiments, the tumor cells are cancer cells, suchas Non-Small-Cell Lung Carcinoma.

The methods of the invention may be thus used for the treatment orprevention of various neoplasia disorders including the group consistingof acral lentiginous melanoma, actinic keratoses, adenocarcinoma,adenoid cycstic carcinoma, adenomas, adenosarcoma, adenosquamouscarcinoma, astrocytic tumors, bartholin gland carcinoma, basal cellcarcinoma, bronchial gland carcinomas, capillary, carcinoids, carcinoma,carcinosarcoma, cavernous, cholangiocarcinoma, chondosarcoma, choriodplexus papilloma/carcinoma, clear cell carcinoma, cystadenoma,endodermal sinus tumor, endometrial hyperplasia, endometrial stromalsarcoma, endometrioid adenocarcinoma, ependymal, epitheloid, Ewing'ssarcoma, fibrolamellar, focal nodular hyperplasia, gastrinoma, germ celltumors, glioblastoma, glucagonoma, hemangiblastomas,hemangioendothelioma, hemangiomas, hepatic adenoma, hepaticadenomatosis, hepatocellular carcinoma, insulinoma, intaepithelialneoplasia, interepithelial squamous cell neoplasia, invasive squamouscell carcinoma, large cell carcinoma, leiomyosarcoma, lentigo malignamelanomas, malignant melanoma, malignant mesothelial tumors,medulloblastoma, medulloepithelioma, melanoma, meningeal, mesothelial,metastatic carcinoma, mucoepidermoid carcinoma, neuroblastoma,neuroepithelial adenocarcinoma nodular melanoma, oat cell carcinoma,oligodendroglial, osteosarcoma, pancreatic polypeptide, papillary serousadenocarcinoma, pineal cell, pituitary tumors, plasmacytoma,pseudosarcoma, pulmonary blastoma, renal cell carcinoma, retinoblastoma,rhabdomyosarcoma, sarcoma, serous carcinoma, small cell carcinoma, softtissue carcinomas, somatostatin-secreting tumor, squamous carcinoma,squamous cell carcinoma, submesothelial, superficial spreading melanoma,undifferentiated carcinoma, uveal melanoma, verrucous carcinoma, vipoma,well differentiated carcinoma, and Wilm's tumor. Accordingly, thecompositions and methods described herein can be used to treat bladdercancer, brain cancer (including intracranial neoplasms such as glioma,meninigloma, neurinoma, and adenoma), breast cancer, colon cancer, lungcancer (SCLC or NSCLC) ovarian cancer, pancreatic cancer, and prostatecancer.

Methods of Making the Compounds of the Invention

The invention also relates to methods of making the compounds andcompositions of the invention. The compounds and compositions can beprepared by any of the applicable techniques of organic synthesis. Manysuch techniques are well known in the art. However, many of the knowntechniques are elaborated in Compendium of Organic Synthetic Methods(John Wiley & Sons, Mew York), Vol. 1, Ian T. Harrison and ShuyenHarrison, 1971; Vol. 2, Ian T. Harrison and Shuyen Harrison, 1974; Vol.3, Louis S. Hegedus and Leroy Wade, 1977; Vol. 4, Leroy G. Wade, Jr.,1980; Vol. 5, Leroy G. Wade, Jr., 1984; and Vol. 6, Michael B. Smith; aswell as standard organic reference texts such as March's AdvancedOrganic Chemistry: Reactions, Mechanisms, and Structure, 5th Ed. by M.B. Smith and J. March (John Wiley & Sons, New York, 2001), ComprehensiveOrganic Synthesis; Selectivity, Strategy & Efficiency in Modern OrganicChemistry, in 9 Volumes, Barry M. Trost, Ed.-in-Chief (Pergamon Press,New York, 1993 printing)); Advanced Organic Chemistry, Part B: Reactionsand Synthesis, Second Edition, Cary and Sundberg (1983); ProtectingGroups in Organic Synthesis, Second Edition, Greene, T. W., and Wutz, P.G. M., John Wiley & Sons, New York; and Comprehensive OrganicTransformations, Larock, R. C., Second Edition, John Wiley & Sons, NewYork (1999).

A number of exemplary methods for the preparation of the compositions ofthe invention are provided below. These methods are intended toillustrate the nature of such preparations are not intended to limit thescope of applicable methods.

Generally, the reaction conditions such as temperature, reaction time,solvents, work-up procedures, and the like, will be chose common in theart for the particular reaction to be performed. The cited referencematerial, together with material cited therein, contains detaileddescriptions of such conditions. Typically the temperatures will be−100° C. to 200° C., solvents will be aprotic or protic depending on theconditions required, and reaction times will be 1 minute to 10 days.Work-up typically consists of quenching any unreacted reagents followedby partition between a water/organic layer system (extraction) andseparation of the layer containing the product.

Oxidation and reduction reactions are typically carried out attemperatures near room temperature (about 20° C.), although for metalhydride reductions frequently the temperature is reduced to 0° C. to−100° C. Heating can also be used when appropriate. Solvents aretypically aprotic for reductions and may be either protic or aprotic foroxidations. Reaction times are adjusted to achieve desired conversions.

Condensation reactions are typically carried out at temperatures nearroom temperature, although for non-equilibrating, kinetically controlledcondensations reduced temperatures (0° C. to −100° C.) are also common.Solvents can be either protic (common in equilibrating reactions) oraprotic (common in kinetically controlled reactions). Standard synthetictechniques such as azeotropic removal of reaction by-products and use ofanhydrous reaction conditions (e.g. inert gas environments) are commonin the art and will be applied when applicable.

Protecting Groups. The term “protecting group”, “blocking group”, or“PG” refers to any group which, when bound to a hydroxy or otherheteroatom prevents undesired reactions from occurring at this group andwhich can be removed by conventional chemical or enzymatic steps toreestablish the hydroxyl group. The particular removable blocking groupemployed is not always critical and preferred removable hydroxylblocking groups include conventional substituents such as, for example,allyl, benzyl, acetyl, chloroacetyl, thiobenzyl, benzylidene, phenacyl,methyl methoxy, silyl ethers (e.g., trimethylsilyl (TMS),t-butyl-diphenylsilyl (TBDPS), or t-butyldimethylsilyl (TBS)) and anyother group that can be introduced chemically onto a hydroxylfunctionality and later selectively removed either by chemical orenzymatic methods in mild conditions compatible with the nature of theproduct. The R groups of Formula (I) can also be protecting groups, asdescribed herein.

Suitable hydroxyl protecting groups are known to those skilled in theart and disclosed in more detail in T. W. Greene, Protecting Groups inOrganic Synthesis; Wiley: New York, 1981 (“Greene”) and the referencescited therein, and Kocienski, Philip J.; Protecting Groups (Georg ThiemeVerlag Stuttgart, New York, 1994), both of which are incorporated hereinby reference.

Protecting groups are available, commonly known and used, and areoptionally used to prevent side reactions with the protected groupduring synthetic procedures, i.e. routes or methods to prepare thecompounds by the methods of the invention. For the most part thedecision as to which groups to protect, when to do so, and the nature ofthe chemical protecting group “PG” will be dependent upon the chemistryof the reaction to be protected against (e.g., acidic, basic, oxidative,reductive or other conditions) and the intended direction of thesynthesis.

Protecting groups do not need to be, and generally are not, the same ifthe compound is substituted with multiple PGs. In general, PG will beused to protect functional groups such as carboxyl, hydroxyl, thio, oramino groups and to thus prevent side reactions or to otherwisefacilitate the synthetic efficiency. The order of deprotection to yieldfree, deprotected groups is dependent upon the intended direction of thesynthesis and the reaction conditions to be encountered, and may occurin any order as determined by the artisan.

Various functional groups of the compounds of the invention may beprotected. For example, protecting groups for —OH groups (whetherhydroxyl, carboxylic acid, or other functions) include “ether- orester-forming groups”. Ether- or ester-forming groups are capable offunctioning as chemical protecting groups in the synthetic schemes setforth herein. However, some hydroxyl and thio protecting groups areneither ether- nor ester-forming groups, as will be understood by thoseskilled in the art. For further detail regarding carboxylic acidprotecting groups and other protecting groups for acids, see Greene,cited above. Such groups include by way of example and not limitation,esters, amides, hydrazides, and the like.

Salts and Solvates

Pharmaceutically acceptable salts of compounds described herein arewithin the scope of the invention and include acid or base additionsalts which retain the desired pharmacological activity and are notbiologically undesirable (e.g., the salt is not unduly toxic,allergenic, or irritating, and is bioavailable). When a compound has abasic group, such as, for example, an amino group, pharmaceuticallyacceptable salts can be formed with inorganic acids (such ashydrochloric acid, hydroboric acid, nitric acid, sulfuric acid, andphosphoric acid), organic acids (e.g. alginate, formic acid, aceticacid, benzoic acid, gluconic acid, fumaric acid, oxalic acid, tartaricacid, lactic acid, maleic acid, citric acid, succinic acid, malic acid,methanesulfonic acid, benzenesulfonic acid, naphthalene sulfonic acid,and p-toluenesulfonic acid) or acidic amino acids (such as aspartic acidand glutamic acid). When the compound of the invention has an acidicgroup, such as for example, a carboxylic acid group, it can form saltswith metals, such as alkali and earth alkali metals (e.g. Na⁺, Li⁺, K⁺,Ca²⁺, Mg²⁺, Zn²⁺), ammonia or organic amines (e.g. dicyclohexylamine,trimethylamine, triethylamine, pyridine, picoline, ethanolamine,diethanolamine, triethanolamine) or basic amino acids (e.g. arginine,lysine and ornithine). Such salts can be prepared in situ duringisolation and purification of the compounds or by separately reactingthe purified compound in its free base or free acid form with a suitableacid or base, respectively, and isolating the salt thus formed.

Many of the molecules disclosed herein contain one or more ionizablegroups [groups from which a proton can be removed (e.g., —COOH) or added(e.g., amines) or which can be quaternized (e.g., amines)]. All possibleionic forms of such molecules and salts thereof are intended to beincluded individually in the disclosure herein. With regard to salts ofthe compounds described herein, one of ordinary skill in the art canselect from among a wide variety of available counterions those that areappropriate for preparation of salts of this invention for a givenapplication. In specific applications, the selection of a given anion orcation for preparation of a salt may result in increased or decreasedsolubility of that salt.

Examples of suitable salts of the compounds described herein includetheir hydrochlorides, hydrobromides, sulfates, methanesulfonates,nitrates, maleates, acetates, citrates, fumarates, tartrates (e.g.,(+)-tartrates, (−)-tartrates or mixtures thereof including racemicmixtures), succinates, benzoates and salts with amino acids such asglutamic acid. These salts may be prepared by methods known to thoseskilled in the art. Also included are base addition salts such assodium, potassium, calcium, ammonium, organic amino, or magnesium salt,or a similar salt. When compounds of the present invention containrelatively basic functionalities, acid addition salts can be obtained bycontacting the neutral form of such compounds with a sufficient amountof the desired acid, either neat or in a suitable inert solvent.Examples of acceptable acid addition salts include those derived frominorganic acids like hydrochloric, hydrobromic, nitric, carbonic,monohydrogencarbonic, phosphoric, monohydrogenphosphoric,dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, orphosphorous acids and the like, as well as the salts derived organicacids like acetic, propionic, isobutyric, maleic, malonic, benzoic,succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Alsoincluded are salts of amino acids such as arginate and the like, andsalts of organic acids like glucuronic or galatunoric acids and thelike. Certain specific compounds of the invention can contain both basicand acidic functionalities that allow the compounds to be converted intoeither base or acid addition salts.

Certain compounds of the invention can exist in unsolvated forms as wellas solvated forms, including hydrated forms. In general, the solvatedforms are equivalent to unsolvated forms and are encompassed within thescope of the invention. Certain compounds of the invention may exist inmultiple crystalline or amorphous forms. In general, all physical formsare equivalent for the uses contemplated by the invention and areintended to be within the scope of the invention.

The terns “solvate” refers to a solid compound that has one or moresolvent molecules associated with its solid structure. Solvates can formwhen a compound is crystallized from a solvent. A solvate forms when oneor more solvent molecules become an integral part of the solidcrystalline matrix upon solidification. The compounds of the formulasdescribed herein can be solvates, for example, ethanol solvates. Anothertype of a solvate is a hydrate. A “hydrate” likewise refers to a solidcompound that has one or more water molecules intimately associated withits solid or crystalline structure at the molecular level. Hydrates canform when a compound is solidified or crystallized in water, where oneor more water molecules become an integral part of the solid crystallinematrix. The compounds of the formulas described herein can be hydrates.

Pharmaceutical Compositions

The following describes information relevant to pharmaceutical andpharmacological embodiments and is further supplemented by informationin the art available to one of ordinary skill. The exact formulation,route of administration and dosage can be chosen by an individualphysician or clinician in view of a patient's condition (see e.g., Finglet al. in The Pharmacological Basis of Therapeutics, 1975, Ch. 1).

It should be noted that the attending physician would know how to andwhen to terminate, interrupt, or adjust administration due to toxicity,or to organ dysfunctions, etc. Conversely, the attending physician wouldalso know to adjust treatment to higher levels if the clinical responsewere not adequate (in light of or precluding toxicity aspects). Themagnitude of an administered dose in the management of the disorder ofinterest can vary with the severity of the condition to be treated andto the route of administration. The severity of the condition may, forexample, be evaluated, in part, by standard prognostic evaluationmethods. Further, the dose and perhaps dose frequency, can also varyaccording to circumstances, e.g. the age, body weight, and response ofthe individual patient. A program comparable to that discussed abovealso may be used in veterinary medicine.

Depending on the specific conditions being treated and the targetingmethod selected, such agents may be formulated and administeredsystemically or locally. Techniques for formulation and administrationmay be found in Alfonso and Gennaro (1995) and elsewhere in the art.

The compounds can be administered to a patient in combination with apharmaceutically acceptable carrier, diluent, or excipient. The phrase“pharmaceutically acceptable” refers to those ligands, materials,compositions, and/or dosage forms that are, within the scope of soundmedical judgment, suitable for use in contact with the tissues of humanbeings and animals without excessive toxicity. Irritation, allergicresponse, or other problem or complication, commensurate with areasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” includes any and allsolvents, dispersion media, diluents, coatings, surfactants,antioxidants, preservatives (e.g., antibacterial agents, antifungalagents), isotonic agents, absorption delaying agents, salts, buffers,preservatives, drugs, drug stabilizers, gels, binders, excipients,disintegration agents, lubricants, sweetening agents, flavoring agents,dyes, such like materials and combinations thereof, as would be known toone of ordinary skill in the art (see, for example, Remington'sPharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp.1289-1329, incorporated herein by reference). Except insofar as anyconventional carrier is incompatible with the active ingredient, its usein the chemotherapeutic or pharmaceutical compositions is contemplated.

A DNQ_(d) or DNQ compound may be combined with different types ofcarriers depending on whether it is to be administered in solid, liquidor aerosol form, and whether it need to be sterile for such routes ofadministration as injection. The present invention can be administeredintravenously, intradermally, intraarterially, intraperitoneally,intralesionally, intracranially, intraarticularly, intraprostaticaly,intrapleurally, intratracheally, intranasally, intravitreally,intravaginally, intrarectally, topically, intratumorally,intramuscularly, intraperitoneally, subcutaneously, subconjunctival,intravesicularlly, mucosally, intrapericardially, intraumbilically,intraocularally, orally, topically, locally, injection, infusion,continuous infusion, localized perfusion bathing target cells directly,via a catheter, via a lavage, in lipid compositions (e.g., liposomes),or by other method or any combination of the forgoing as would be knownto one of ordinary skill in the art (see, for example, Remington'sPharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990,incorporated herein by reference).

The actual dosage amount of a composition of the present inventionadministered to a patient can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient and on the route ofadministration. The practitioner responsible for administration will, inany event, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

When administered to a subject, effective amounts will depend, ofcourse, on the particular cancer being treated; the genotype of thespecific cancer; the severity of the cancer; individual patientparameters including age, physical condition, size and weight,concurrent treatment, frequency of treatment, and the mode ofadministration. These factors are well known to the physician and can beaddressed with no more than routine experimentation. In someembodiments. It is preferred to use the highest safe dose according tosound medical judgment.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of a DNQ_(d) or DNQ compound. In otherembodiments, the an active compound may comprise between about 2% toabout 75% of the weight of the unit, or between about 25% to about 60%,for example, and any range derivable therein. In other non-limitingexamples, a dose may also comprise from about 0.1 mg/kg/body weight, 0.5mg/kg/body weight, 1 mg/kg/body weight, about 5 mg/kg/body weight, about10 mg/kg/body weight, about 20 mg/kg/body weight, about 30 mg/kg/bodyweight, about 40 mg/kg/body weight, about 50 mg/kg/body weight, about 75mg/kg/body weight, about 100 mg/kg/body weight, about 200 mg/kg/bodyweight, about 350 mg/kg/body weight, about 500 mg/kg/body weight, about750 mg/kg/body weight, to about 1000 mg/kg/body weight or more peradministration, and any range derivable therein. In non-limitingexamples of a derivable range from the numbers listed herein, a range ofabout 10 mg/kg/body weight to about 100 mg/kg/body weight, etc., can beadministered, based on the numbers described above.

In any case, the composition may comprise various antioxidants to retardoxidation of one or more component. Additionally, the prevention of theaction of microorganisms can be brought about by preservatives such asvarious antibacterial and antifungal agents, including, but not limitedto parabens (e.g., methylparabens, propylparabens), chlorobutanol,phenol, sorbic acid, thimerosal or combinations thereof.

A DNQ_(d) or DNQ compound may be formulated into a composition in a freebase, neutral or salt form. Pharmaceutically acceptable salts includethe salts formed with the free carboxyl groups derived from inorganicbases such as for example, sodium, potassium, ammonium, calcium orferric hydroxides; or such organic bases as isopropylamine,triethylamine, histidine or procaine.

Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate.

Dragee cores are optionally provided with suitable coatings. For thispurpose, concentrated sugar solutions may be used, which may optionallycontain gum arable, talc, polyvinyl pyrrolidone, carbopol gel,polyethylene glycol, and/or titanium dioxide, lacquer solutions, andsuitable organic solvents or solvent mixtures. Dyestuffs or pigments maybe added to the tablets or dragee coatings for identification or tocharacterize different combinations of active compound doses.

In embodiments where the composition is in a liquid form, a carrier canbe a solvent or dispersion medium comprising, but not limited to, water,ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethyleneglycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes)and combinations thereof. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin; by the maintenanceof the required particle size by dispersion in carriers such as, forexample liquid polyol or lipids; by the use of surfactants such as, forexample hydroxypropylcellulose (HPC); or combinations thereof suchmethods. In many cases, it will be preferable to include isotonicagents, such as, for example, sugars, sodium chloride or combinationsthereof.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount of the appropriate solvent with variousother ingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thevarious sterilized active ingredients into a sterile vehicle whichcontains the basic dispersion medium and/or the other ingredients. Inthe case of sterile powders for the preparation of sterile injectablesolutions, suspensions or emulsion, the preferred methods of preparationare vacuum-drying or freeze-drying techniques which yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered liquid medium thereof. The liquid mediumshould be suitably buffered if necessary and the liquid diluent firstrendered isotonic prior to injection with sufficient saline or glucose.

The composition should be stable under the conditions of manufacture andstorage, and preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. Thus, preferred compositionshave a pH greater than about 5, preferably from about 5 to about 8, morepreferably from about 5 to about 7. It will be appreciated thatendotoxin contamination should be kept minimally at a safe level, forexample, less that 0.5 ng/mg protein.

In particular embodiments, prolonged absorption of an injectablecomposition can be brought about by the use in the compositions ofagents delaying absorption, such as, for example, aluminum monostearate,gelatin or combinations thereof.

Formulation of DNQ Compounds for in Vivo Administration

The aqueous solubility of DNQ at pH 7.4 In phosphate buffered saline(PBS) was measured by LC-MS. DNQ was sonicated for 30 minutes in PBSthen undissolved solid was removed by filtration through a 0.45 μmsyringe filter and the filtrate was analyzed by LC-MS (λ=275 nm, ESI-TOFin negative mode). The optimal sonication time was determined bysonicating DNQ for 1, 5, 10, and 30 minutes. While the concentration ofDNQ in solution increased substantially between 1, 5, and 10 minutes,there was only a minor difference between 10 and 30 minutes. During the30 minute sonication the water bath warmed to 45° C. (samples werecooled to room temperature before filtration). A calibration curve wasgenerated from 1-100 μM by dissolving DNQ in methanol to a concentrationof 500 μM and making dilutions of this stock in 80:20 wafer:methanol.The calibration curve (measure by UV absorbance) was linear over thisrange; 1 μM was approximately the limit of detection. The solubility ofDNQ in PBS was measured to be 115 μM. The solution was very pale yellow.

Because of the poor aqueous solubility of DNQ we investigated the use of2-hydroxypropyl-beta-cyclodextrln (HPβCD), a common excipient, toimprove the solubility of DNQ. In the absence of HPβCD, the solubilityof DNQ increases significantly in strongly basic solutions and DNQprecipitates when the pH is returned to neutral. However, in thepresence of a sufficient amount of HPβCD, DNQ does not precipitate whenthe phi is returned to neutral. This same neutral solution of DNQ inHPβCD cannot be made directly (i.e. without pH adjustment). Thisindicates that DNQ compounds deprotonate in base and this deprotonatedmolecule forms a tight complex with HPβCD which is stable enough toprevent protonation as the pH decreases. The only proton on DNQ thatmight reasonably be deprotonated in aqueous base is the N—H. Althoughthe acidity of the N—H bond of DNQ has not been measured. It has beenmeasured for a derivative of DNQ and found to have a pKa of 8.0.

The protocol for formulating DNQ compounds in HPβCD is as follows: theDNQ compound is slurried in a 20% solution of HPβCD in pH 7.4 PBS andthe pH is then increased by the addition of 10 M NaOH to inducedissolution of the DNQ compound. The pH is returned to pH 7.5-8.0 by thecareful addition of 1 M HCl. A 3.3 μM solution of the DNQ compound canbe made by this method which is stable at least 24 hours. Thisrepresents a 30-fold increase in solubility of DNQ over PBS alone.

We initially chose a 20% HPβCD solution. However, we have found thatβ-lap was formulated as a 40% solution of HPβCD for human clinicaltrials and our experience with DNQ indicates that the concentration ofDNQ increases linearly with that of HPβCD; thus a 40% HPβCD solutionwould permit the creation of a 6.6 mM solution of DNQ and other DNQcompounds.

Combination Therapy

Active ingredients described herein (e.g., compounds of Formula (I)) canalso be used in combination with other active ingredients. Suchcombinations are selected based on the condition to be treated,cross-reactivities of ingredients and pharmaco-properties of thecombination. For example, when treating cancer, the compositions can becombined with other anti-cancer compounds (such as paclitaxel orrapamycin).

It is also possible to combine a compound of the invention with one ormore other active ingredients in a unitary dosage form for simultaneousor sequential administration to a patient. The combination therapy maybe administered as a simultaneous or sequential regimen. Whenadministered sequentially, the combination may be administered in two ormore administrations.

The combination therapy may provide “synergy” and “synergistic”, i.e.the effect achieved when the active ingredients used together is greaterthan the sum of the effects that results from using the compoundsseparately. A synergistic effect may be attained when the activeingredients are: (1) co-formulated and administered or deliveredsimultaneously in a combined formulation; (2) delivered by alternationor in parallel as separate formulations; or (3) by some other regimen.When delivered in alternation therapy, a synergistic effect may beattained when the compounds are administered or delivered sequentially,e.g. in separate tablets, pills or capsules, or by different injectionsin separate syringes. In general, during alternation therapy, aneffective dosage of each active ingredient is administered sequentially,i.e. serially, whereas in combination therapy, effective dosages of twoor more active ingredients are administered together. A synergisticanti-cancer effect denotes an anti-cancer effect that is greater thanthe predicted purely additive effects of the individual compounds of thecombination.

Combination therapy is further described by U.S. Pat. No. 6,833,373(McKearn et al.), which includes additional active agents that can becombined with the compounds described herein, and additional types ofcancer and other conditions that can be treated with a compounddescribed herein.

Accordingly, it is an aspect of this invention that a DNQ_(d) or DNQ canbe used in combination with another agent or therapy method, preferablyanother cancer treatment. A DNQ_(d) or DNQ may precede or follow theother agent treatment by intervals ranging from minutes to weeks. Inembodiments where the other agent and expression construct are appliedseparately to the cell, one would generally ensure that a significantperiod of time did not elapse between the time of each delivery, suchthat the agent and expression construct would still be able to exert anadvantageously combined effect on the cell. For example, in suchinstances, it is contemplated that one may contact the cell, tissue ororganism with two, three, four or more modalities substantiallysimultaneously (i.e., within less than about a minute) with the organicarsenical. In other aspects, one or more agents may be administeredwithin about 1 minute, about 5 minutes, about 10 minutes, about 20minutes about 30 minutes, about 45 minutes, about 60 minutes, about 2hours, about 3 hours, about 4 hours, about 6 hours, about 8 hours, about9 hours, about 12 hours, about 15 hours, about 18 hours, about 21 hours,about 24 hours, about 28 hours, about 31 hours, about 35 hours, about 38hours, about 42 hours, about 45 hours, to about 48 hours or more priorto and/or after administering the organic arsenical. In certain otherembodiments, an agent may be administered within from about 1 day, about2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 8days, about 9 days, about 12 days, about 15 days, about 16 days, about18 days, about 20 days, to about 21 days prior to and/or afteradministering the organic arsenical. In some situations, it may bedesirable to extend the time period for treatment significantly,however, where several weeks (e.g., about 1, about 2, about 3, about 4,about 6, or about 8 weeks or more) lapse between the respectiveadministrations.

Administration of the chemotherapeutic compositions of the presentinvention to a patient will follow general protocols for theadministration of chemotherapeutics, taking into account the toxicity,if any. It is expected that the treatment cycles would be repeated asnecessary. It also is contemplated that various standard therapies oradjunct cancer therapies, as well as surgical intervention, may beapplied in combination with the described arsenical agent. Thesetherapies include but are not limited to chemotherapy, radiotherapy,immunotherapy, gene therapy and surgery.

Chemotherapy

Cancer therapies can also include a variety of combination therapieswith both chemical and radiation based treatments. Combinationchemotherapies include the use of chemotherapeutic agents such as,cisplatin, etoposide, irinotecan, camptostar, topotecan, paciltaxel,docetaxel, epothilones, taxotere, taxol, tamoxifen, 5-fluorouracil,methoxtrexate, temozolomide, cyclophosphamide, SCH 66336, R115777,L778,123, BMS 214662, IRESSA™ (gefitinib), TARCEVA™ (erlotinibhydrochloride), antibodies to EGFR, GLEEVEC™ (imatinib), intron, ara-C,adriamycin, cytoxan, gemcitabine, uracil mustard, chlormethine,ifosfamide, melphalan, chlorambucil, pipobroman, triethylenemelamine,triethylenethiophosphoramine, busulfan, carmustine, lomustine,streptozocin, dacarbazine, floxuridine, cytarabine, 6-mercaptopurine,6-thioguanine, fludarabine phosphate, pentostatine, vinblastine,vincristine, vindesine, bleomycin, doxorubicin, dactinomycin,daunorubicin, epirubicin, idarubicin, mithramycin, deoxycoformycin,Mitomycin-C, L-Asparaginase, Teniposide 17α-Ethinylestradiol,Diethylstilbestrol, testosterone, prednisone, fluoxymesterone,dromostanolone propionate, testolactone, megestrolacetate,methylprednisolone, methyltestosterone, prednisolone, triamcinolone,chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine,medroxyprogesteroneacetate, leuprolide, flutamide, toremifene,goserelin, carboplatin, hydroxyurea, amsacrine, procarbazine, mitotane,mitoxantrone, levamisole, navelbene, anastrazole, letrazole,capecitabine, reloxafine, droloxafine, hexamethylmelamine, Avastin,herceptin, Bexxar, Velcade, Zevalin, Trisenox, Xeloda, Vinorelbine,Porfimer, Erbitux™ (cetuximab), Liposomal, Thiotepa, Altretamine,Melphalan, Trastuzumab, Lerozole, Fulvestrant, Exemestane, Fulvestrant,Ifosfomide, Rituximab, C225, Campath, carboplatin, procarbazine,mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan,chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin,doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP 16),tamoxifen, raloxifene, estrogen receptor binding agents, taxol(paclitaxel), gemcitabine, navelbine, farnesyl-protein transferaseinhibitors, transplatinum, 5-fluorouracil, vincristine, vinblastine andmethotrexate, or any analog or derivative variant of the foregoing.

Radiotherapy

Other factors that cause DMA damage and have been used extensivelyinclude what are commonly known as gamma.-rays, X-rays, and/or thedirected delivery of radioisotopes to tumor cells. Other forms of DMAdamaging factors are also contemplated such as microwaves andUV-irradiation. It is most likely that all of these factors affect abroad range of damage on DNA, on the precursors of DMA, on thereplication and repair of DNA, and on the assembly and maintenance ofchromosomes. Dosage ranges for X-rays range from dally doses of 50 to200 roentgens for prolonged periods of time (3 to 4 wk), to single dosesof 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely,and depend on the half-life of the isotope, the strength and type ofradiation emitted, and the uptake by the neoplastic cells. The terms“contacted” and “exposed,” when applied to a cell, are used herein todescribe the process by which a therapeutic construct and achemotherapeutic or radiotherapeutic agent are delivered to a targetcell or are placed in direct juxtaposition with the target cell. Toachieve cell killing or stasis, both agents are delivered to a cell in acombined amount effective to kill the cell or prevent it from dividing.

Immunotherapy

Immunotherapeutics, generally, rely on the use of immune effector cellsand molecules to target and destroy cancer cells. The immune effectormay be, for example, an antibody specific for some marker on the surfaceof a tumor cell. The antibody alone may serve as an effector of therapyor it may recruit other cells to actually affect cell killing. Theantibody also may be conjugated to a drug or toxin (chemotherapeutic,radionucleotide, ricin A chain, cholera toxin, pertussis toxin, etc.)and serve merely as a targeting agent. Alternatively, the effector maybe a lymphocyte carrying a surface molecule that interacts, eitherdirectly or indirectly, with a tumor cell target. Various effector cellsinclude cytotoxic T cells and NK cells.

Immunotherapy, thus, could be used as part of a combined therapy, inconjunction with gene therapy. The general approach for combined therapyis discussed below. Generally, the tumor cell must bear some marker thatis amenable to targeting, i.e., is not present on the majority of othercells. Many tumor markers exist and any of these may be suitable fortargeting in the context of the present invention. Common tumor markersinclude carcinoembryonic antigen, prostate specific antigen, urinarytumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72,HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, lamininreceptor, erb B and p155.

Gene Therapy

In yet another embodiment, the secondary treatment is a secondary genetherapy in which a therapeutic polynucleotide is administered before,after, or at the same time a first chemotherapeutic agent. Delivery ofthe chemotherapeutic agent in conjunction with a vector encoding a geneproduct will have a combined anti-hydroproliferative effect on targettissues.

Surgery

Approximately 60% of persons with cancer will undergo surgery of sometype, which includes preventative, diagnostic or staging, curative andpalliative surgery. Curative surgery is a cancer treatment that may beused in conjunction with other therapies, such as the treatment of thepresent invention, chemotherapy, radiotherapy, hormonal therapy, genetherapy, immunotherapy and/or alternative therapies. Curative surgeryincludes resection in which all or part of cancerous tissue isphysically removed, excised, and/or destroyed. Tumor resection refers tophysical removal of at least part of a tumor, in addition to tumorresection, treatment by surgery includes laser surgery, cryosurgery,electrosurgery, and microscopically controlled surgery (Mohs' surgery).It is further contemplated that the present invention may be used inconjunction with removal of superficial cancers, precancers, orincidental amounts of normal tissue.

One of ordinary skill in the art will appreciate that startingmaterials, biological materials, reagents, synthetic methods,purification methods, analytical methods, assay methods, and biologicalmethods other than those specifically exemplified can be employed in thepractice of the invention without resort to undue experimentation. Allart-known functional equivalents, of any such materials and methods areintended to be included in this invention. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and there is no intention that in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims.

The following Examples are intended to illustrate the above inventionand should not be construed as to narrow its scope. One skilled in theart will readily recognize that the Examples suggest many other ways inwhich the invention could be practiced. It should be understood thatnumerous variations and modifications may be made while remaining withinthe scope of the invention. The invention may be further understood bythe following non-limiting examples.

Abbreviations Used in the Schemes and Examples may Include the Following

-   A549=adenocarcinoma human alveolar basal epithelial cells-   ATP=adenosine triphosphate-   β-lap=β-lapachone-   DHE=dihydroethidium-   DNQ=Deoxynyboquinone-   DNQ_(d)=Any analog or derivative of deoxynyboquinone-   ELISA=enzyme-linked immunosorbent assay-   h=hour(s)-   H596=[NCl-HS596] human lung adenosquamous carcinoma cell line-   HT1080=primate fibrosarcoma cell line-   LD₅₀=lethal dose having 50% probability of causing death-   LD₉₀=lethal dose having 90% probability of causing death-   LD₁₀₀=lethal dose having 100% probability of causing death-   MCF-7=human breast adenocarcinoma cell line-   MDA-MB-231=human breast cancer cell line-   MIA-PaCa2=Pancreatic cancer cell line-   mins=minute(s)-   NADH=nicotinamide adenine dinucleotide-   NQO1=NAD(P)H:quinone oxidoreductase 1-   NSCLC=non-small-cell lung cancer cells-   OCR=oxygen consumption rates-   p53=a tumor suppressor protein-   PC-3=human prostate cancer cell line-   ROS=reactive oxygen species-   ±SE=standard error-   siRNA=small interfering ribonucleic acid-   shRNA=small hairpin ribonucleic acid-   μM=micromolar-   nM=nanomolar-   μmol=micromole

EXAMPLES Example 1 Preparation of SCH 538415 and DNQ

A. Synthesis of SCH 538415. Of the potential precursors todiazaanthracenols evaluated, the most successful route was through 2-61(Scheme 1.12), The specific disposition of halides in compound 2-61 wasenvisioned to arise through sequential directed ortho lithiation of2,6-dichloroanisole. As diiodination under such conditions is difficult.Intermediate disilane 2-62 was targeted. Chloride is known to be a weakdirecting group for ortho-lithiation, but has been successfully utilizedin a number of settings. However, lithium-chloride exchange appeared tobe the dominant reaction in a variety of n- and s-butyllithium-mediatedreactions, presumably due to the strong directing effects of the methoxygroup. It was found that deprotonation with lithium diisopropyl amide(LDA) was both efficient and selective for the 3- and 5-positions(Scheme 1.12). In addition, the two-step sequence could be carried outin one pot. Trimethylsilyl chloride was an effective in situ-quenchreagent, with highest conversions when additions of reagent weresequential, beginning with LDA. Iododesilylation of 2-62 by the actionof iodine monochloride was rapid and quantitative, producing 2-61.

Miyaura borylation conditions then provided cross-coupling partner 2-64in good yield but contaminated with variable amounts ofbis(pinacolborane). The two-step yield after the subsequentcross-coupling was higher when 2-64 was used without purification, thusafter workup 2-64 was typically taken directly into the cross-coupling.The double Suzuki cross-coupling of 2-64 with iodoamide 2-17 proceededas expected with an acceptable 55% yield of 2-65. Finally, thePd(OAc)₂/X-Phos system effectively cyclized the electron-rich,ortho-substituted compound 2-65 in excellent yield.

After a brief survey of oxidants, it was found that, though moreelectron rich than diazaanthracene 2-51, 2-53 was still challenging tooxidize. Attempts to deprotect the phenol, which should be more prone tooxidation than the anisole, met with failure. Attempts to follow thissynthetic scheme beginning with other protecting groups (e.g., —OMOM,—OBn, —OPBM, —OTHP, —OSEM) surprisingly all failed at various steps. Wefound that oxidation of 2-53 was possible under forcing conditions.Thus, brief heating of 2-53 in concentrated nitric acid produced SCH538415 as a bright red-orange solid in 40% yield, along with variableamounts (0-14%) of nitrated product 2-66, the structure of which wasconfirmed by single-crystal x-ray diffraction. By this route the firsttotal synthesis of the natural product was completed in six steps and9.7% overall yield from 2,6-dichloroanisole. Spectral data matched thatof the natural product.

B. Synthesis of DNQ. The simplest approach to apply this route to thesynthesis of DNQ would involve a selective mono-N-methylation ofnor-methyl diazaanthracenol 2-67 (Scheme 1.13). Primary amide 2-70 wassynthesized but found to be an unreactive partner in the Suzuki couplingwith 2-64. The N-para-methoxybenzyl amide 2-71 was then synthesized in82% yield from 2-butynoic acid by hydroiodination and treatment of thecorresponding acid chloride with p-methoxybenzyl amine (Scheme 1.13).This route was employed because the reaction of ethyl 2-butynoate withp-methoxybenzyl amine resulted primarily in 1,4-addition to the alkyne.The Suzuki cross-coupling of 2-64 with 2-71 produced 2-72 in 42% yield.The subsequent amidation proceeded in quantitative yield to generate theprotected tricyclic compound 2-73. Removal of the PMB protecting groupsfrom 2-73 in hot concentrated HBr was rapid and produced 2-67 in 97%yield. We found that methylation of 2-67 was highly unselective,generating a mixture of mono- and di-N- and O-alkylated products.Although a trace amount of DNQ was isolated by subsequent hydrolysis ofo-alkylated products, oxidation in nitric acid, and chromatographicpurification, we deemed this route impractical.

An alternate route to DNQ focused on the synthesis of nonsymmetricdiamide 2-74 (Scheme 1.14). Although routes involving iterative Suzukicoupling initially appeared promising, a mixed cross-coupling betweenbisboronate 2-64 and iodoamides 2-17 and 2-71 was found to be thesimplest method to form 2-74. Separation of 2-74 from the accompanyingsymmetric products was easily effected by chromatography. Aryl amidationunder the previously employed conditions efficiently formed tricycle2-75 along with variable amounts of unprotected amide 2-76. Isolation atthis step was unnecessary, as subjection of the crude amidation productsto acidic hydrolysis produced 2-77 in 76% yield over two steps.Oxidation of phenol 2-77 was markedly more facile than oxidation ofanisole 2-53. Oxidation of 2-77 catalyzed by salcomine under O₂ producedDNQ in 77% yield. Overall the synthesis consisted of 7 steps in thelongest linear sequence, 12% overall yield. This compares to 11 stepsand <0.84% yield for the previous synthesis of DNQ (Rinehart et al. J.Am. Chem. Soc. 1961, 83, 3729; Forbis et al., J. Am. Chem. Soc., 1973,95, 5(503). By this method, 400 mg of DNQ has been synthesized in asingle sequence without encountering any difficulty related to scale.

C. Synthesis of nor-methyl anthraquinone 2-78. Having previouslysynthesized 2-73 we sought to make the previously-reported nor-methylDNQ derivative 2-78 for comparison with DNQ and SCH 538415. Unlike for2-52, heating in HBr over 4 hours also removes the phenol-protectingmethyl group from 2-73. Oxidation of diazaanthracenol 2-79 in HNO₃ at60° C. generates a red solution from which a red-orange solidprecipitates upon cooling and dilution with water. Compound 2-78 wasinsoluble in all solvents evaluated except concentrated acids or bases.

In conclusion, the total syntheses of SCH 538415, DNQ, anddeoxynybomycin were accomplished using concise and modular routes thatrelied heavily on modern Pd-mediated cross-coupling reactions. Threesynthetic hurdles were overcome: formation of the tricyclic skeleton,oxidation of the diazaanthracene to the anthraquinone, and synthesis ofnon-symmetric intermediates en route to DNQ. The tricycle was formedthrough a double intramolecular amidation of electron-rich andsterically hindered aryl chlorides which had to be carried through fromthe beginning of the sequence. Attempts to oxidize diazaanthracene 2-51were unsuccessful, but the more electron rich anisole 2-53 wassuccessfully oxidized to SCH 538415 under forcing conditions. It wasfound that a straight-forward mixed Suzuki cross-coupling was a viablemethod for producing the non-symmetric diamide 2-74 en route to DNQ.Finally, oxidation of phenol 2-77 was accomplished under mild,cobalt-catalyzed conditions to deliver the target compound, DNQ.

Materials and Methods. Reagents were purchased from Aldrich, Strem(metal catalysts and ligands), GFS (alkynes), Frontier Scientific(B₂pin₂) and used without further purification unless otherwise noted.Elesclomol and tirapazamine were synthesized according to the literatureprocedures (U.S. Patent Publication No. 2003/0195258 (Koya et al.); andFuchs et al. J Org Chem 2001, 66, 107, respectively). Solvents weredried by passage through columns packed with activated alumina (THF,CH₂Cl₂, diethyl ether) or activated molecular sieves (DMSO). Amines werefreshly distilled over CaH₂ under a nitrogen atmosphere. Reactionsinvolving n-BuLi or LDA were performed using standard Schlenk techniquesunder argon.

¹H-NMR and ¹³C-NMR spectra were recorded on Varian Unity spectrometersat 500 MHz and 125 MHz, respectively. Spectra generated from a solutionof CDCl₃ were referenced to residual chloroform (¹H: δ 7.26 ppm, ¹³C: δ77.23 ppm). Spectra generated in mixtures of CDCl₃ and CD₃OD werereferenced to tetramethylsilane (¹H: δ 0.00 ppm; or CD₃OD (¹³C: δ 49.0ppm). Spectra generated from d-TFA were referenced to residual H (¹H: δ11.50 ppm) or F₃CCO₂D (¹³C: δ 164.2 ppm).

Preparation of LDA: To an oven-dried Schlenk flask was added dry THF(23.2 mL), diisopropylamine (14.0 mL, 99.9 mmol) and a stir bar. Aftercooling the flask in a dry ice/isopropanol bath, n-BuLi (1.6 M inhexanes, 62.8 mL, 100 mmol) was added dropwise over about 15 minutes.The flask was then transferred to an ice bath for temporary storage.

To an oven-dried 250 mL Schlenk flask and stir bar under argon was addeddry THF (50 mL) and 2,6-dichloroanisole (2-63, 500 mL, 36.5 mmol). Theflask was chilled in a −75° C. bath and freshly prepared LDA (1.0 M, 44mL, 44 mmol) was added by syringe over 15 minutes. The reaction wasstirred for 3.25 h between −65 and −75° C. and was kept in thistemperature range for the duration of the reaction. TMSCl (5.60 mL, 43.8mmol) was then added and the mixture was stirred for 1 h. A secondaliquot of LDA (47 mL, 47 mmol) was then added followed, after threemore hours, by a second aliquot of TMSCl (5.50 mL, 50.9 mmol). Themixture was allowed to stir and warm overnight and had reached −20° C.after 14 h. The cloudy yellow solution was quenched with 15 mL water,which dissolved the precipitate. The mixture was transferred to aseparatory funnel, diluted with 1M HCl (15 mL) and ether (20 mL), andshaken and separated. The aqueous phase was extracted once with ether(1×30 mL), and the combined organic extracts were washed with brine(1×20 mL), dried over MgSO₄, filtered, and evaporated to a pale yellowoil which solidified under vacuum. Recrystallization from MeOH/H₂O intwo crops yielded 7.25 and 1.42 g of 2-62 as white flakes (8.67 g, 27.0mmol, 74%).

¹H-NMR (CDCl₃, 500 MHz):

7.25 (s, 1H, aryl CH), 3.89 (s, 3H, OCH₃), 0.36 (s, 18H, Si(CH₃)₃.¹³C-NMR (CDCl₃, 125 MHz, d1 delay set to 20 sec, dm=nny):

151.9, 138.7, 137.0, 136.7, 60.5, −0.5. HRMS (EI) calcd for C₁₃H₂₂OSi₂Cl(M)⁺: 320.0586, found: 320.0583. Melting point: 78.5-80.5° C. IR (cm⁻¹,thin film in CDCl₃): 2958 (w), 1397 (w), 1342 (m), 1251 (m).

Iodine monochloride (11.41 g, 70.28 mmol) was weighed into a 100 mLround-bottom flask with a stir bar and dissolved in 35 mL CH₂Cl₂. Theflask was chilled on an ice-water bath and 2-62 (7.49 g, 23.3 mmol) wasadded portion-wise at a slow enough rate to keep the solutiontemperature below 20° C. After the addition, a thick precipitate formedand more CH₂C₂ (35 mL) was added to dissolve it. Stirring was continuedfor 0.5 h, after which water (10 mL) was added and NaHSO₃ was added byspatula until the color of the solution ceased to fade. The aqueouslayer was a clear yellow and the organic layer was colorless. Themixture was poured into a separatory funnel, diluted with water (20 mL)and CH₂Cl₂ (50 mL), and shaken and separated. The aqueous phase wasfurther extracted with CH₂Cl₂ (2×15 mL). The combined organic extractswere then washed with brine (1×20 mL), dried over MgSO₄, filtered andevaporated to yield 2-61 as a white solid (9.965 g, 23.24 mmol, 99.6%).Product may be recrystallized from hexanes to produce colorless needles.

¹H-NMR (CDCl₃, 500 MHz):

8.14 (s, aryl CH), 3.88 (s, —OCH₃). ¹³C-NMR (CDCl₃, 125 MHz):

152.6, 144.3, 135.0, 97.7, 60.8. HRMS (EI) calcd for C₇H₆OCl₂ (M)⁺:427.7729, found: 427.7731. Melting point: 138.5-140.5° C. IR (cm⁻¹, thinfilm in CCl₄): 1528 (w), 1394 (m), 1361 (s), 1005 (s).

To a hot, oven-dried Schlenk flask with stir bar was added KOAc (1.24 g,12.6 mmol) and vacuum was applied to dry the salt until the flask wascool. To this flask was added PdCl₂(dppf) (0.256 g, 15.0 mol %), B₂pin₂(1.17 g, 4.59 mmol) and 2-61 (0.896 g, 2.09 mmol). The flask wasevacuated and backfilled with argon (3×). Dry DMSO (12 mL) was added bysyringe and the mixture was plunged into an oil bath at 80° C. Colorchanged from yellow to red to black over five minutes. The mixture wasallowed to stir with heating for 19 hours. After cooling, the mixturewas poured into a separatory funnel with an additional 5 mL DMSO andthen extracted with hexanes (3×100 mL). The combined hexane extractswere washed with water (2×100 mL) until the organic layer was clear. Theorganic fraction was then dried over MgSO₄, filtered, and evaporated toan off-white solid. The crude solid (0.858 g) was a mixture of 6 andB₂pin₂ (molar ratio ˜3:1). The yield of 2-64 was calculated from thisratio to be 79% (712 mg, 1.66 mmol). This material was generally usedwithout purification in the subsequent Suzuki cross couplings.Recrystallization of the mixture from pentane yields pure 2-64 ascolorless needles.

¹H-NMR (CDCl₃, 500 MHz):

7.67 (s, 1H, aryl CH), 3.86 (s, 3H, OCH₃), 1.37 (s, 24H, C(CH₃)₂).¹³C-NMR (CDCl₃, 125 MHz):

152.3, 138.0, 137.1 (2C), 130 (broad, 2C), 84.5 (2C), 60.5, 25.0 (8C).HRMS (ESI-TOF) calcd for C₁₉H₂₈B₂O₅Cl₂ (M+H)⁺: 429.1587, found:429.1591. Melting point: 185-187° C. IR (cm⁻¹, thin film in CCl₄): 2982(m), 2935 (w), 1574 (m), 1446 (m), 1362 (s), 1333 (s), 1143 (s), 1033(m).

To a Schlenk flask with a stir bar was added crude 2-64 (404 mg totalmass, estimated 323 mg and 0.755 mmol 2-64), PdCl₂(dppf) (123 mg, 20 mol%), K₂CO₃ (627 mg, 4.54 mmol), and 2-17 (510 mg, 2.27 mmol) and theflask was evacuated and backfilled with argon three times. Water (1 mL)and DME (9 mL) were added by syringe after degassing the solvents bybubbling with argon for 45 minutes. The flask was plunged into an oilbath at 80° C. for 3 h. The mixture was poured into a separatory funneland diluted with water (5 mL). The mixture was extracted with EtOAc(3×20 mL). The combined organic extracts were dried over MgSO₄, filteredand evaporated to a deep red oil. The crude product was dissolved inCH₂Cl₂ and loaded onto a silica plug (diameter: 48 mm, height: 30 mm)and eluted with EtOAc. The fractions containing 2-65 were evaporated toa red solid. This solid was stirred in 3 mL EtOAc and filtered. Thesolid was washed twice with 2 mL portions of EtOAc then allowed to dryon the filter. Compound 2-65 was isolated as colorless crystals (152 mg,0.409 mmol, 55% from estimated 2-64, 43% over two steps from 2-61).

¹H-NMR (CDCl₃, 500 MHz):

6.73 (s, 1H, aryl CH), 5.94 (q, 2H, J=1.5 Hz, vinylic CH), 5.59 (bs, 2H,NH), 3.91 (s, 3H, OCH₃), 2.69 (d, 6H, J=4.5 Hz, NCH₃), 2.12 (d, 6H,allylic CH₃, J=1.0 Hz). ¹³C-NMR (CDCl₃, 125 MHz):

165.8, 145.4, 140.0, 125.5, 124.2, 124.2, 123.3, 61.0, 26.5, 25.4. HRMS(ESI-TOF) calcd for C₁₇H₂₀N₂O₃Cl₂ (M+H)⁺: 371.0929, found: 371.0915.Melting point: Darkened at 210° C., decomposed at 230° C. IR (cm⁻¹, thinfilm in CDCl₃): 3383 (w), 3326 (b, w), 2979 (w), 2939 (w), 1663 (m),1641 (m), 1608 (m), 1525 (m), 1368 (m).

To a 1-dram vial with a stir bar was added 2-65 (16.3 mg, 0.0440 mmol),K₂CO₃ (36.70 mg, 0.266 mmol), Pd/X-Phos (3.25 mg, 10.0 mol %), andX-Phos (2.20 mg, 10.5 mol %). The vial was cycled between vacuum andargon three times and i-PrOH (1.75 mL) was added while flushing the vialwith argon. The vial was sealed with a Teflon-lined cap and immersed inan oil bath at 110° C. The reaction was allowed to stir with heating for16 hours, insoluble materials were removed by filtration through Celiteand rinsed with CH₂Cl₂. The filtrate was evaporated and the residue waspurified by column chromatography (0-5% MeOH in EtOAc). Compound 2-53(12.6 mg, 0.0422 mmol, 96%) was isolated as white solid.

¹H-NMR (CDCl₃, 500 MHz):

7.68 (s, 1H, aryl CH), 6.57 (d, 2H, J=1.5 Hz, vinyl CH), 3.86 (s, 6H,N—CH₃), 3.50 (s, 3H, OCH₃), 2.50 (d, 6H, J=1.0 Hz, allylic CH₃). ¹³C-NMR(CDCl₃, 125 MHz):

164.1, 146.1, 137.2, 136.3, 120.8, 119.8, 116.9, 62.4, 35.9, 19.2. HRMS(ESI-TOF) calcd for C₁₇H₁₈N₂O₃ (M+H)⁺: 299.1396, found: 299.1385.Melting point: >250° C. IR (cm⁻¹, thin film in CDCl₃): 2962 (w), 1648(s), 1619 (m), 1583 (s), 1445 (m), 1394 (m), 1354 (m), 1325 (m), 1035(m).

In a 7-mL vial with a stir bar, 2-53 (29.8 mg, 0.100 mmol) was dissolvedin cone. HNO₃ (3 mL) and the vial was plunged into an oil bath at 80° C.After 15 minutes the flask was removed from heat and the solution wasdiluted with water (4 mL), transferred to a separatory funnel andextracted with CH₂C₂ (3×10 mL). The aqueous phase was carefullyneutralized with 10 M NaOH (a dark color persists when the acid isconsumed) and extracted once more with CH₂Cl₂ (10 mL). The organicextracts were loaded directly onto a silica gel column and eluted with agradient of 0 to 5% MeOH in EtOAc. Product was isolated as a deep redfraction which was evaporated to a bright red-orange solid. This wasdissolved in 5 mL CH₂Cl₂ and filtered to remove a sparingly solubleyellow material. The filtrate was evaporated to yield SCH 538415 as abright red-orange solid (11.9 mg, 0.0399 mmol, 40%). Spectral data matchthe reported natural product SCH 538415 can be further purified bysublimation (180° C., 300 mtorr).

¹H-NMR for 2-10 (CDCl₃, 500 MHz):

6.65 (d, 2H, J=1.0 Hz, vinyl CH), 3.73 (s, 6H, NCH₃), 2.56 (d, 6H, J=1.0Hz, allylic CH₃). ¹³C-NMR for 2-10 (CDCl₃, 125 MHz):

181.5, 178.9, 161.5, 149.1, 143.0, 126.6, 117.0, 34.2, 22.8. HRMS for2-10 (ESI-TOF) calcd for C₁₆H₁₄N₂O₄ (M+H)⁺: 299.1032, found: 299.1020.Melting point for 2-10: >250° C. IR for 2-10 (cm⁻¹, thin film in CDCl₃):1662 (b, w), 1362 (w). ¹H-NMR for 2-66 (CDCl₃, 500 MHz):

6.70 (d, 1H, J=1.0 Hz, vinyl CH), 3.83 (s, 3H, NCH₃), 3.74 (s, 3H,NCH₃), 2.56 (s: accidental overlap—two separate signals are seen inother spectra, 6H, allylic CH₃). ¹³C-NMR for 2-66 (CDCl₃, 125 MHz):

180.4, 177.8, 161.2, 154.3, 148.9, 142.9, 142.5, 140.8, 127.2, 117.8,115.4, 35.3, 34.2, 22.7, 16.9.

To a 7 mL vial with stir bar was added ethyl 2-butynoate (506 mg, 4.51mmol) and concentrated NH₄OH (˜30%, 1.5 mL, 18 mmol) and the biphasicsolution was stirred vigorously for 17 h during which time a thick whiteprecipitate formed. This solid was collected by filtration and driedunder vacuum to yield 2-69 as a white solid (198 mg, 2.38 mmol, 53%).

¹H-NMR (d⁶-DMSO, 500 MHz):

7.813 (bs, 1H, NH), 7.362 (bs, 1H, NH), 1.91 (s, 3H, CH₃). ¹³C-NMR(d⁶-DMSO, 125 MHz):

154.4, 82.4, 75.9, 3.0. HRMS (EI) calcd for C₄H₅NO (M)⁺: 83.0371, found:83.0370. MP: 151-152.5° C. IR (cm⁻¹, thin film in CDCl₃): 1665 (w), 1583(w), 1369 (w).

To a 1-dram vial with a stir bar was added 2-69 (1.18 mg, 1.41 mmol),Mai (337 mg, 2.25 mmol), and acetic acid (0.52 mL, 9.1 mmol). The vialwas closed with a screw-on cap and plunged into a preheated oil bath at100° C. for 16 h. The deep red reaction mixture was diluted with water(20 mL) and EtOAc (20 mL), treated with NaHSO₃ until the color stoppedfading (pale yellow), then neutralized with 8 mL 1M NaOH. This mixturewas poured into a separatory funnel and the aqueous layer was extractedwith EtOAc (1×10 mL). The combined organic layers were washed with sat.NaHCO₃ (1×10 mL) and brine (1×10 mL), dried over MgSO₄, and evaporatedto yield 162 mg (0.765 mmol, 54%) of 2-70 as a white flakey solid.

¹H-NMR (CDCl₃, 500 MHz):

6.28 (d, 1H, J=1.0 Hz, vinyl CH), 5.80 (bs, 2H, NH₂), 2.69 (d, 3H, J=1.0Hz, allylic CH₃). ¹³C-NMR (CDCl₃, 100 MHz):

165.4, 128.7, 105.7, 353. HRMS (ESI-TOF) calcd for C₄H₆NOI (M+H)⁺:211.9572, found: 211.9582. Melting point: 87-92° C. IR (cm⁻¹, thinfilm): 3332 (s), 3167 (s), 1665 (s), 1622 (s), 1425 (m), 1403 (s), 1260(m).

To a flask containing 2-butynoic acid (0.503 g, 5.98 mmol), NaI (1.434g, 9.57 mmol) and a stir bar was added glacial acetic acid (2.2 mL, 38mmol) and the flask was immersed in an oil bath at 115° C. for 3 h. Thereaction mixture was poured into a separatory flask and diluted withwater (10 mL) and ether (10 mL). NaHSO₃ was added until the color of thesolution faded to a pale yellow, then the mixture was shaken andseparated. The aqueous fraction was extracted with ether (3×10 mL) thenthe combined organic fractions were dried over MgSO₄ and evaporated toyield 2-73 as a white solid (1.19 g, 5.59 mmol, 94%). Acid 2-73 can berecrystallized with good recovery from CHCl₃/heptane to yield largeprismatic crystals.

¹H-NMR (CDCl₃, 500 MHz):

12.19 (bs, 1H, COOH), 6.36 (d, 1H, J=1.5 Hz, vinyl CH), 2.77 (d, 3H,J=1.0 Hz, allylic CH₃). ¹³C-NMR (CDCl₃, 125 MHz):

169.8, 125.4, 117.1, 37.3. HRMS (ESI-TOF) calcd for C₄H₅O₂I (M+Na)⁺:234.9232, found: 234.9234. Melting point: 113-115° C. IR (cm⁻¹, thinfilm in CDCl₃): 2978 (b, m), 2702 (m), 2589 (m), 2506 (m), 1699 (s),1619 (s), 1434 (m), 1406 (m), 1303 (m), 1222 (s).

To an oven-dried 40 mL I-Chem vial with a stir bar was added 2-73 (1.03g, 4.85 mmol) and the flask was evacuated and backfilled with argon. DryCH₂Cl₂ (12 mL) was added and the solution was chilled on an ice-waterbath. Oxalyl chloride (1.25 mL, 14.3 mmol) was added by syringe and thecold bath was removed. After 5 h at room temperature the volatilecomponents were evaporated directly from the vial. Dry CH₂Cl₂ (10 mL)was added to the residual oil and the vial was chilled on a dryice/isopropanol bath. Freshly distilled p-methoxybenzyl amine (740 mg,5.39 mmol) was added dropwise by syringe followed by NEt₃ (0.675 mL,4.85 mmol). The mixture was allowed to warm to −40° C. after 2 h, then 1M HCl (20 mL) was added and the solution was poured into a separatoryfunnel with CH₂Cl₂ (10 mL). Shaken and separated. The aqueous fractionwas extracted with CH₂Cl₂ (4×10 mL) then dried over MgSO₄ and evaporatedto a white solid. The solid was slurried in 50 mL ether and hot filteredthrough Celite. The filtrate was diluted with 50 mL hexanes and heatedto reflux until crystallization began. After cooling, pale tan crystalsof 2-71 were collected by filtration (1.23 g). The mother liquor aspurified by column chromatography (hexanes/EtOAc=2/1) thenrecrystallized from ether/hexanes to yield additional product as whiteneedles (169 mg). Total 2-71: 1.40 g, 4.24 mmol, 87%.

¹H-NMR (CDCl₃, 500 MHz):

7.20 (d, 2H, J=8.0 Hz, aryl CH), 6.82 (d, 2H, J=8.0 Hz, aryl CH), 6.35(bs, 1H, NH), 6.23 (q, 1H, J=1.5 Hz, vinylic CH), 4.37 (d, 2H, J=3.5 Hz,NCH₂), 3.75 (s, 3H, OCH₃), 2.61 (d, 3H, J=1.5 Hz, allylic CH₃). ¹³C-NMR(CDCl₃, 125 MHz):

164.7, 159.1, 130.0, 129.5, 128.9, 114.1, 106.5, 55.4, 43.1, 35.9. HRMS(ESI-TOF) calcd for C₁₂H₁₃NO₂I (M+H)⁺: 332.0148, found: 332.0154.Melting point: 95-98° C. IR (cm⁻¹, thin film in CDCl₃): 3436 (w), 3311(bw), 2959 (w), 2838 (w), 1656 (m), 1513 (s), 1465 (w), 1302 (w), 1250(m), 1176 (m), 1088 (w), 1035 (m).

To a 7-mL vial with a stir bar was added crude 2-64 (97 mg total mass,estimated 81 mg and 0.19 mmol 2-64), PdCl₂(dppf) (30.6 mg, 0.038 mmol,20 mol %), K₂CO₃ (156 mg, 1.13 mmol), and 2-71 (155 mg, 0.468 mmol) andthe vial was evacuated and backfilled with argon three times. Water (1mL) and DME (9 mL) were added by syringe after degassing the solvents bybubbling with argon for 45 minutes. The flask was plunged into an oilbath at 87° C. for 2 h. The mixture was poured info a separatory funneland diluted with water (5 mL). The mixture was extracted with EtOAc (2×5mL). The combined organic extracts were dried over MgSO₄, filtered andevaporated to a deep red oil. The crude product was dissolved in CH₂Cl₂and purified by silica gel chromatography. The product was furtherpurified by recrystallization from chloroform/heptanes. Compound 2-72was isolated a microcrystalline solid (57.4 mg, 0.098 mmol, 42% over twosteps from 2-61).

¹H-NMR (CDCl₃, 500 MHz):

6.99 (bs, 4H), 6.76-6.74 (m, 5H), 5.97 (d, 2H, J=1.5 Hz, vinylic CH),6.0-5.3 (bd, 2H), 4.3-4.1. (bd, 4H), 3.75 (s, 6H, OCH₃), 3.72 (s, 3H,OCH₃), 2.09 (bs, 6H, allylic CH₃). ¹³C-NMR (CDCl₃, 125 MHz):

165.1, 1.59 (bs), 145.3, 140 (bs), 131 (bs), 129.0, 125.6, 124.3, 123.5(bs), 114.0, 60.8, 55.5, 42.9, 25.5.

To a 1-dram vial with 3 stir bar was added 2-72 (29.4 mg, 0.0504 mmol),K₂CO₃ (42.2 mg, 0.305 mmol), and Pd/X-Phos (6.1 mg, 0.005 mmol, 10.0 mol%). The vial was cycled between vacuum and argon and i-PrOH (1.5 mL) wasadded while flushing the vial with argon. The vial was sealed with aTeflon-lined cap and immersed in an oil bath at 85° C. The reaction wasallowed to stir with heating for 14.5 hours. The solvent was evaporatedand the residue was purified by column chromatography (0-100% EtOAc inhexanes). Compound 2-73 (23 mg, quant.) was isolated an oily solid.

¹H-NMR (CDCl₃, 500 MHz):

7.62 (s, 1H, aryl CH), 7.01 (bd, 4H, J=7.5 Hz), 6.74 (d, 4H, J=8.0 Hz)6.58 (s, 2H, vinyl CH), 5.9-5.4 (bs, 2H), 5.2-5.4 (bd, 4H), 3.72 (s, 6H,OCH₃), 2.9 (bs, 3H), 2.48 (s, 6H, a allylic CH₃).

2-73 (200 mg, 0.390 mmol) was slurried in 48% HBr (8 mL) and heated inan oil bath at 115° C. for 7 minutes. The reaction was diluted to 50 mLwith water and the product was collected by filtration and washing withwater and EtOAc. 2-67 was collected as a light yellow solid (102 mg,0.378 mmol, 97%). ¹H-NMR (2:1 CDCl₃:CD₃OD, 500 MHz): δ 7.82 (s, 1H, arylCH), 6.51 (d, 2H, J=1.5 Hz, vinyl CH), 3.94 (s, 3H), 2.59 (d, 6H, J=1.5Hz, allylic CH₃).

To a 40 mL I-Chem vial was added crude 2-64 (750 mg, 1.45 mmol),PdCl₂(dppf) (239.1 mg, 20 mol %), K₂CO₃ (120 g, 8.69 mmol), 2-17 (490.3mg, 2.18 mmol), and 2-71 (625.4 mg, 1.89 mmol) and the vial was cappedwith a screw-on lid with a septum and evacuated and backfilled withargon. Water (1 mL) and DME (10 mL) were added by syringe afterdegassing the solvents by bubbling with argon for 30 minutes. The flaskwas immersed in an oil bath at 90° C. for 2.5 hours. The mixture wasallowed to cool and then was poured into a separatory funnel, dilutedwith water (25 mL) and extracted with EtOAc (3×20 mL). The combinedorganic extracts were washed with brine (1×15 mL), dried over MgSO₄,filtered and evaporated. The residue was purified by columnchromatography (4:1 EtOAc:hexanes) to yield 2-74 as a pale yellow foam(231 mg, 0.484 mmol, 34% yield from estimated 2-64, 27% over two stepsfrom 2-61).

¹H-NMR (CDCl₃, 500 MHz):

7.03 (bs, 2H, PMB CH), 6.74 (d, 3H, J=8.0 Hz, PMB CH and aryl CH), 6.55(bs, 1H, NH), 5.99 (bs, 1H, NH), 5.96 (s, 1H, vinyl CH), 5.92 (s, 1H,vinyl CH), 4.20 (bd, 2H, J=29 Hz, benzyl CH₂), 3.79 (s, 3H), 3.73 (s,3H), 2.57 (d, 3H, J=3.5 Hz, NCH₃), 2.09 (s, 3H, allyl CH₃), 2.07 (bs,3H, allyl CH₃). ¹H-NMR (CDCl₃, 60° C., 500 MHz)

7.02 (bd, 2H, J=6.5 Hz, PMB CH), 6.75 (d, 2H, J=8.0 Hz, PMB CH), 6.74(s, 1H, aryl CH), 6.3 (bs, 1H, NH), 5.97 (s, 1H, vinyl CH), 5.93 (s, 1H,vinyl CH), 5.87 (bs, 1H, NH), 4.21 (bs, 2H, benzyl CH₂), 3.80 (s, 3H),3.73 (s, 3H), 2.58 (d, 3H, J=4.5 Hz, NCH₃), 2.09 (s, 6H, allyl CH₃),2.06 (s, 3H, allyl CH₃). ¹³C-NMR (CDCl₃, 60° C., 125 MHz):

165.9, 165.1, 159.1, 153.5, 145.4, 144.6, 140.2, 140.1, 131.1, 128.9,125.6, 125.5, 124.5, 124.3, 123.5, 114.2, 60.7, 55.4, 42.9, 26.3, 25.2(2C). HRMS (ESI-TOF) calcd for C₂₄H₂₆Cl₂N₂O₄ (M+H)⁺: 477.1348, found:477.1331. Melting point: 157-162° C. IR (cm⁻¹, thin film in CDCl₃): 3404(w), 2940 (w), 1663 (w), 1613 (w), 1514 (m), 1369 (w), 1249 (w), 1029(w).

To a 1-dram vial with a stir bar was added 2-74 (39.9 mg, 0.0835 mmol),K₂CO₃ (69.9 mg, 0.506 mmol), Pd/X-Phos (6.15 mg, 10.0 mol %), and X-Phos(4.03 mg, 10.1 mol %). The vial was cycled between vacuum and argonthree times and i-PrOH (3.4 mL) was added while flushing the vial withargon. The vial was sealed with a Teflon-lined cap and immersed in anoil bath at 110° C. The reaction was allowed to stir with heating for 15hours. After cooling, insoluble materials were removed by filtrationthrough Celite and rinsing with CH₂Cl₂. At this point 2-75 can beisolated in >95% yield by chromatographic purification with 0 to 2.5%MeOH in EtOAc.

¹H-NMR (CDCl₃, 500 MHz):

7.64 (s, 1H, anisole CH), 7.09 (d, 2H, J=8.5 Hz, PMB CH), 6.73 (d, 2H,J=8.5 Hz, PMB CH), 6.65 (d, 1H, J=1.0 Hz, vinyl CH), 6.52 (d, 1H, J=1.0Hz, vinyl CH), 5.30 (bd, 2H, benzyl CH₂), 3.72 (s, 3H), 3.65 (s, 3H),3.25 (bs, 3H), 2.52 (d, 3H, J=1.0 Hz, allylic CH₃), 2.46 (d, 3H, J=1.5Hz, allylic CH₃).] The filtrate was evaporated and a stir bar and 2 mL48% HBr were added. The flask was immersed in an oil bath at 110° C.[After 5 minutes of heating, 2-76 can be isolated in >95% yield bydilution of the acid in water and collection of the resultingprecipitate. ¹H-NMR (2:1 CDCl₃:CD₃OD, 500 MHz):

7.84 (s, 1H, aryl CH), 6.58 (d, 1H, J=1.5 Hz, vinyl CH), 6.55 (d, 1H,J=1.5 Hz, vinyl CH), 3.98 (s, 3H), 3.82 (s, 3H), 2.61 (d, 3H, J=1.0 Hz,allylic CH₃), 2.57 (d, 3H, J=1.5 Hz, allylic CH₃). ¹³C-NMR (CDCl₃, 125MHz):

164.7, 164.0, 149.9, 148.0, 134.7, 134.2, 133.9, 120.5, 120.1, 119.7,117.8, 117.6, 62.8, 34.2, 19.5, 19.1. HRMS (ESI-TOF) calcd forC₁₆H₁₆N₂O₃ (M+H)⁺: 285.1239, found: 285.1226].

After 17 hours the reaction was removed from heat. The mixture wascarefully rendered basic over an ice bath by adding 10 M NaOH until theprecipitate dissolved into a yellow solution. The residual solid wasremoved by filtration through hardened filter paper and discarded. Thefiltrate was rendered acidic with 1 M HCl, whereupon a colloidalprecipitate formed. The mixture was then centrifuged (3220×g for 5minutes). The resulting semi-compact gelatinous solid was collected byfiltration through hardened filter paper and dried to a constant massunder vacuum to yield 17.2 mg of 2-77 as light brown solid (17.2 mg,0.0636 mmol, 76% over 3 steps).

¹H-NMR (d-TFA, 500 MHz):

8.3 (s, 1H, aryl CH), 7.20 (s, 1H, vinyl CH), 7.03 (s, 1H, vinyl CH),4.38 (s, 3H, NCH₃), 2.78 (s, 3H, allylic CH₃), 2.75 (s, 3H, allylicCH₃). ¹³C-NMR (d-TFA, 125 MHz):

167.2, 166.6, 160.4, 159.3, 135.7, 134.9, 133.6, 125.2, 123.6, 120.4,119.9, 116.1, 39.6, 20.9, 20.2. HRMS (ESI-TOF) calcd for C₁₅H₁₄N₂O₃(M+H)⁺: 271.1083, found: 271.1076. Melting point: >250° C. IR (cm⁻¹,thin film in CDCl₃): 3156 (b, m), 1674 (s), 1651 (s), 1589 (m), 1562(m), 1432 (w), 1378 (m), 1351 (m), 1332 (m), 1052 (w).

To a 7-mL vial containing 2-77 (17.2 mg, 0.0636 mmol), salcomine (2.08mg, 0.0064 mmol), and a stir bar was added MeOH (4 mL) and CH₂Cl₂ (2mL). A balloon containing O₂ was fitted over the mouth of the vial andthe slurry was stirred at room temperature. The solid dissolved afterabout 30 minutes. After 2.75 h stirring, the solvent was evaporated andthe residue was purified by silica gel chromatography (6% MeOH inCH₃Cl₂). DNQ was collected as a bright pink-red solid (13.9 mg, 0.0489mmol, 77%). Product can be further purified by sublimation (200° C., 300mtorr).

¹H-NMR (d⁵-pyridine, 500 MHz):

6.81 (s, 1H, vinyl CH), 6.76 (s, 1H, vinyl CH), 4.97 (bs, NH), 3.99 (s,3H, NCH₃), 2.57 (d, 3H, allylic CH₃), 2.53 (d, 3H, allylic CH₃). HRMS(ESI-TOF) calcd for C₁₅H₁₂N₂O₄ (M+H)⁺: 285.0875, found: 285.0864.Melting point: >250° C. IR (cm⁻¹, thin film in CDCl₃): 1656 (b, w), 1585(w), 1469 (w), 139 (w), 1379 (w), 1344 (w), 1290 (w), 1098 (w).

Example 2 Analysis of DNQ and DNQ Derivatives

TABLE 1 Properties of Various Compounds and Compositions of theinvention. C: K: Fold F: CH₂Cl₂: Protection D: NQO1 H: I: J: MeOH ofMCF7 NQO1 V_(max) G: DMSO CH₂Cl₂ THF 2:1 L: B: cells w/ Initial E:(μmol/min/ Catalytic solu- solu- solu- solu- Binding A: MCF7 IC₅₀ 25 μMVelocity @ NQO1 K_(m) μmol Efficiency bility bility bility bility EnergyID (μM) dicoumarol 1 μM (μM) protein) (M⁻¹*s⁻¹) (mM) (mM) (mM) (mM)(kcal/mol) DNQ 0.19 7.8 1900 ± 100 1.0 ± 0.1 4000 ± 100 6.7E+07 3.1 0.70.42 8.5 14 SCH 3.9 3.5 32 ± 2 10.6 ± 0.4  402 ± 6  6.3E+05 15 25 3.7 608 87 0.25 6.1 1900 ± 200 1.2 ± 0.1 4400 ± 100 6.1E+07 110 180 34 140 17251 0.27 5.5 2100 ± 300 0.89 ± 0.12 4200 ± 200 7.9E+07 10 7.6 6.1 30 18107 0.28 4.5 1400 ± 100 2.1 ± 0.2 4200 ± 100 3.3E+07 31 5.5 4.7 37 28109 0.53 4.5 1500 ± 100 2.7 ± 0.2 5000 ± 200 3.1E+07 24 9.5 18 19 14 1290.77 2.8 1300 ± 200 1.4 ± 0.1 3000 ± 100 3.6E+07 14 24 17 75 18 253 0.932.8  600 ± 100 3.6 ± 0.2 4400 ± 100 1.4E+07 10 6.1 4.6 18 21 255 3.4 1.6250* 6.4* 1900* 4.2 2 10 271 5 1.2 250 ± 20 3.9 ± 0.5 1070 ± 80  4.6E+0621 10 6.2 46 14 131 6.3 1.1 350 ± 10 4.2 ± 0.6 1600  10 17 12 49 10 3736 0.9 75 ± 4 1.5 ± 0.3 200 ± 20 2.2E+06 7.1 35 17 64 4 127 8 ND 320 ±60 1.4 ± 0.1 750 ± 20 8.9E+06 1.2 34 19 34 32 9-253 0.11 ± 0.04 9 1700 ±100 1.9 ± 0.1 5600 ± 200 4.9E+07 16 23 20 100 10 9-281 0.79 ± 0.1  41600 ± 200 2.7 ± 0.1 6300 ± 100 3.9E+07 24 0.3 0.67 11 10 9-251 0.19 ±0.02 8 2000 ± 100 1.2 ± 0.2 4800 ± 200 6.7E+07 15 31 23 120 16 9-2490.15 ± 0.03 12 2100 ± 90  1.1 ± 0.1 4800 ± 200 7.3E+07 8.6 9.7 8.8 58 17DNQ-2 0.12 ± 0.01 10 1590 ± 70  1.5 ± 0.2 4100 ± 200 4.6E+07 8.9 3.9 3.432 20 (10-41) 9-255 0.19 ± 0.01 11 1700 ± 40  1.3 ± 0.1 3900 ± 1005.0E+07 17 22 16 95 12 9-257 0.26 ± 0.04 11 1850 ± 60  1.0 ± 0.1 3500 ±100 5.8E+07 18 32 27 140 13 2-77  0.22 ± 0.01 6 1260 ± 40  2.8 ± 0.24700 ± 100 2.8E+07 25 13 8.1 51 13 2-99  0.23 ± 0.02 9 970 ± 20 3.58 ±0.08 4320 ± 40  2.0E+07 21 5.6 7.4 46 11 10-47  16 11 3.9 35 14 8-2553.4 1.6 280 ± 40 4.4 ± 0.6 1540 ± 80  5.9E+06 4.2 2 10 19 10-53  <0.11.5 1.8 5.9 24 10-51  <0.1 1.8 1.8 6.4 21 2-97  0.83 ± 0.13 5  700 ± 2003.1 ± 0.3 3200 ± 100 1.7E+07 71 120 87 79 30 β-lap 3.7 2.1 150  170 1650660 1400 MMC 14 1 RH1 0.21 0.85

Example 3 Antitumor Effect in A549 Lung Cancer Xenograft in Nude Miceand Potentiation of Radiation

A formulation of DNQ in HPβCD was sufficiently concentrated to dose atreasonable levels in mice (˜10 mg/kg from a 200 μL injection). We areactively experimenting with DNQ in mouse models of cancer. The Boothmanlab has completed a pilot study of DNQ in an A549 subcutaneous xenograftmodel in nude mice (Blanco et al., Cancer Res. 2010, 70, 3896). Becauseβ-lap has been shown to potentiate the effect of ionizing radiation, andbecause DNQ and β-lap have similar mechanisms of cytotoxicity, mice weretreated with or without ionizing radiation to determine if DNQ alsopotentiates the effect of radiation (FIG. 3).

Athymic nude mice were inoculated with A549 NSCLC cells and establishedtumors were allowed to grow to ˜200 mm³. Mice were assigned to one ofsix treatment groups—four mice per group. Three groups received DNQ (0,2.5, or 5 mg/kg) by tail vein injection on days 1, 3, 5, 7, and 9. Thedoses of DNQ were chosen based on the observation that 10 mg/kg DNQelicited adverse reactions in mice. The other three groups received thesame DNQ regimen and also received 2 grays of ionizing radiation on eachtreatment day. Tumor volumes were measured over the subsequent weeks(FIG. 3).

Because each treatment group comprised only four animals, thestatistical significance of the results is low. However, an examinationof the average trends in FIG. 3 indicates that both groups that received5 mg/kg DNQ experienced a slower the rate of tumor growth relative tocontrol. With these exciting preliminary results in hand, mouse modelsof cancer with full-size treatment groups are being pursued.

The maximum tolerated dose (MTD) of DNQ in mice contained a similarconcentration of HPβCD to the MTD of β-lap. Although HPβCD is toleratedat very high doses by IP injection (>5 g/kg), the MTD of HPβCD by IVinjection for mice has not been reported. The MTD of HPβCD by IVinjection in rats has been reported to be approximately 2.25 g/kg (Gouldand Scott, Food Chem. Toxicol. 2005, 43, 1451). If the MTD in mice ismuch lower than that, it is possible that the vehicle is causing thedose-limiting toxicity.

Because of the difficulty of repeated tall vein injections in mice, ourgroup is using IP administration of compound. The MTD of HPβCD appearsto be greater than can be delivered even by a 1 mL injection of asaturated aqueous solution of HPβCD. Thus, when injected IP, the dose ofHPβCD will likely be irrelevant. We have found the MTD of DNQ to beabout 5 mg/kg by IP injection. At this dose mice become lethargic andunresponsive to touch. The effects subside after 1-2 hours and the micereturn to a healthy state. Dally injections of this dose were nottolerated, but dosing every other day was tolerated.

Although the mouse model experiment provided encouraging results, twoliabilities for future use of DNQ were noted: 1) DNQ is not welltolerated by mice at the doses required to see an anticancer effect, and2) the low aqueous solubility of DNQ necessitates the use of HPβCD, theuse of which is undesirable from both cost and potential toxicitystandpoints.

Example 4 Synthesis and Evaluation of Derivatives of DNQ

Limitations of DNQ for in vivo administration. Although DNQ displayedpromising antitumor efficacy in the preliminary murine cancer model, weforesaw two potential hurdles for the future administration of DNQ invivo: low aqueous solubility, and a narrow therapeutic window. We wereconfident that an analysis of the structure-activity relationship (SAR)of DNQ, enabled by the synthesis of a library of derivatives, wouldreveal compounds that are both more soluble than and equipotent to DNQ.We planned to determine the MTD of all such derivatives in mice.Assuming that equivalent cytotoxicity in cell culture would translateinto equivalent antitumor doses in mice, the compound with the highestMTD would provide the widest therapeutic window. Thus, we set out todiscover a new lead compound by 1) synthesizing a library of DNQderivatives, 2) establishing the SAR of DNQ, 3) establishing thestructure-solubility relationship of DNQ, and 41 determining the MTD ofthe best new derivatives in healthy mice. By this process, a new leadcompound would be identified and its efficacy in multiple mouse modelsof cancer would be explored.

Poor aqueous solubility. Despite possessing multiple sites for hydrogenbonding interactions with water, DNQ is poorly soluble (100 μM in pH 7.4PBS buffer). Two potential reasons for the insolubility of DNQ are itsability to π-stack in the solid state and double intermolecular hydrogenbonding. Both of these effects are clearly shown in a crystal structureof DNQ (FIG. 4) recently reported by Li and coworkers who isolated DNQfrom a strain of Pseudonocardia isolated from the South China Sea (Li etal., Mar. Drugs 2011, 9, 1428). Because SCH 538415 is less potent thanDNQ, breaking up the intermolecular hydrogen bonding by substitution atthe N—H might produce inactive derivatives. Therefore focus was placedon disruption of the π-stacking with the thought that breaking up thisπ-stacking by the addition of freely-rotating short alkyl chains wouldimprove aqueous solubility despite increasing the lipophilicity.

The synthetic route to DNQ was expected to be amenable to substitutionat the three positions indicated by colored spheres in FIG. 5. Althoughit was expected that the aqueous solubility of DNQ derivatives bearingshort alkyl chains would be greater than that of DNQ, the lipophilicityof sufficiently long appendages would overcome the advantage ofdisrupting the π-stacking and result in less soluble derivatives. Theoptimal alkyl length for maximal aqueous solubility would be determinedby synthesizing a series of n-alkyl derivatives and assessing theirproperties. The addition of polar functional groups (e.g., —OH, —NH,—COOH, etc.) to DNQ would be expected to have a strong positive effecton aqueous solubility.

Improving solubility in HPβCD solutions. Like DNQ, β-lap suffers frompoor aqueous solubility (˜160 μM in pH 7.4 PBS). It was found thatdissolution of β-lap in a solution of HPβCD resulted in ˜150-foldincrease in solubility (20% HPβCD in H₂O). When DNQ was dissolved inHPβCD solution, solubility increased only 30-fold. The difference infold solubility increase between DNQ and β-lap is ascribed to thefollowing: whereas β-lap is insoluble because of its high lipophilicity(which is offset by complexation with HPβCD) DNQ is insoluble because ofstable solid packing—on which HPβCD has no effect. The same DNQderivatives described in the previous section would likely benefit moreby dissolution with HPβCD than DNQ itself—by virtue of their increasedlipophilicity. Furthermore, certain appendages might display very highaffinity for HPβCD which would result in significant solubility gains.Thus, the solubility of all derivatives, whether more or less soluble inwater than DNQ, would be tested in the presence of HPβCD.

Improving solubility in organic solvents, Another way that β-lap hasbeen administered in vivo is encapsulated in micelles (Blanco et al.,Cancer Res. 2010, 70, 3896). Whereas liposomal formulations are usefulfor water-soluble drugs—trapping them in the aqueous interior of theliposome—micelles trap lipophilic drugs in their hydrophobic interior.Thus, proposed lipophilic derivative of DNQ might be good candidates formicellar formulations. It was expected that the solubility of DNQderivatives in organic solvents—such as DCM and THF—would be orders ofmagnitude greater than the solubility of DNQ itself because of thecombined effects of destabilizing π-stacking and increasinglipophilicity.

Predicted SAR. A large number of compounds with diverse structures havebeen identified as substrates of NQO1 (Colucci et al., Org. Biomol.Chem. 2008, 6, 637). This promiscuity suggests that derivatization ofDNQ could yield compounds that are also substrates for the enzyme.Molecular modeling based on crystal structures of NQO1 support thisnotion. FIG. 6 (left) shows NQO1 with the inhibitor dicoumarol bound inthe active site (Asher et al., Biochemistry 2006, 45, 6372). As drawn,the western coumarin of DIC π-stacks with the FAD cofactor, while theeastern half extends down a cavity that opens into the active site. Sidechains of DNQ should be able to access this cavity as well. Northeast ofthe active site is another cavity through which the FAD passes thatcould potentially allow for substrate access as well. The western halfof the active site is closed off—any side chains forced to lie in thatarea would reduce the binding affinity of the compound. Molecularmodeling of DNQ in the NQO1 active site suggests that DNQ will π-stackwith the FAD isoalloxazine moiety (FIG. 6, right), but that theorientation of DNQ in the binding pocket may not be important. Theaffinity of side chains of DNQ derivatives for the active site may makefor stronger substrate-enzyme binding. Compounds that bind too weakly toNQO1 would be poor substrates, but compounds that bind too tightly wouldbe inhibitor. Thus compounds with optimal binding properties should bethe most potent cytotoxins in cell culture.

A preliminary set of nine derivatives was to be synthesized to probe theeffect of substituents on DNQ at the three positions noted (Scheme 4.1).These compounds would be assessed for their potency in cell culture andtheir dependence on NQO1 for cytotoxicity by co-treating withdicoumarol. Based on the NQO1-dependent cytotoxicity of this initial setof derivatives, a second, larger set would be synthesized to furtherexplore the SAR. Compounds that are equipotent to DNQ would then beassessed for their solubility properties in water, in HPβCD, and inorganic solvents.

Synthesis and NQO1-dependent cytotoxicity of an initial set of 9 DNQderivatives. We synthesized a set of nine derivatives bearing ethyl,propyl, or undecyl chains at each of three positions that are easilymodified by the existing synthetic route to DNQ (Scheme 4.1). Therequired alkyne esters (of the type 4-1 for derivatives 4-8 and 4-9),alkyne acids (of the type 4-3 for derivatives 4-11 and 4-12), andprimary amines (of the type 4-2 for derivatives 4-14 through 4-16) werecommercially available and were used to make the indicated compoundsthrough routes analogous to that for DNQ. The alkynes required tosynthesize derivatives 4-10 and 4-13 were synthesized from alkyne 4-17as shown in Scheme 4.2.

The NQO1-dependent cytotoxicity of the first set of derivatives wasdetermined in MCF-7 cells which express high levels of NQO1. Cells wereexposed to DNQ for 2 hours in the presence or absence of 25 μM DIC. Themedia was then removed and cells were washed once with media then freshmedia was added and the cells were incubated for 72 hours. Cell deathwas assessed using the sulforhodamine B assay previously described. IC₅₀values for these compounds are listed in Table 4.1. While ethylderivative 4-8 was equipotent with DNQ, propyl derivative 4-9 wassignificantly less toxic. Dodecyl derivative 4-10 was insoluble in DMSO(<0.1 mM) and could not be assessed in this assay. We found thatsubstitution opposite the NH (4-11 and 4-12) was poorly tolerated, witheven a single methylene addition causing a significant loss of toxicity.As was the case with 4-10, compound 4-13 was insoluble in DMSO.Substitution off the nitrogen appeared well tolerated (4-14 and 4-15).Dodecyl derivative 4-16 was inactive and the protective effect of DICcould not be assessed because of its poor solubility.

As further evidence that the activity of these DNQ derivatives isdependent on their ability to be reduced by NQO1, full enzymaticmeasurements in vitro were performed. The results are shown in Table4.1. We found a strong correlation between catalytic efficiency ofsubstrates in vitro and their cell culture toxicity. The most activederivatives in cell culture displayed catalytic efficiencies above 3×10⁷M⁻¹S⁻¹. This provides strong evidence that the cytotoxicity of DNQ andits derivatives are dependent primarily upon the activity of NQO1 withinthe cell.

TABLE 4.1 Cytotoxicity and NQO1 dependence of nine initial DNQderivatives. IC₅₀ vs. MCF-7 cells Fold (

 M ± s.e.) protection k_(cat)/K_(M) Compound +25 

 M DIC by DIC (M⁻¹s⁻¹ × 10⁷) DNQ 0.13 ± 0.02 1.7 ± 0.3 14 6.7 4-8  0.28± 0.06 1.3 ± 0.4 4.5 3.3 4-9  0.90 ± 0.25 2.6 ± 0.6 2.8 1.4 4-10 —^(a)—^(a) —^(a) —^(a) 4-11 0.55 ± 0.06 4.9 ± 0.8 8.9 2.3 4-12 3.4 ± 0.6 5.3± 1.2 1.6 0.59 4-13 —^(a) —^(a) —^(a) —^(a) 4-14 0.20 ± 0.01 3.4 ± 0.217 3.1 4-15 0.13 ± 0.01 1.3 ± 0.4 10 4.6 4-16 8.0 ± 1.6 —^(a) —^(a) 0.89^(a)Insufficiently soluble to determine.

Synthesis and NQO1-dependent cytotoxicity of a second set of DNQderivatives. Having concluded that substitution off the nitrogen waslikely to produce active derivatives, efforts were made to synthesize asecond set of derivatives (Scheme 4.3) to determine the optimal lengthof an n-alkyl chain (4-22 through 4-25) as well as the effect ofmultiple substitutions (4-26 through 4-29), branched alkyl substituents(4-30 through 4-39), and polar functionalities (4-40 through 4-42).Deviations from the standard synthetic route are outlined below.

Synthesis of a second set of DNQ derivatives. Whereas most of thederivatives were synthesized by following the same protocol used tosynthesize DNQ, a few derivatives required minor modifications of theroute. The most sterically hindered amines were slow to react withethyl-2-butynoate to generate the corresponding alkynyl amides,resulting in poor yields. It was found that the desired amides could bemore efficiently synthesized using the route designed for the synthesisof the PMB-protected amide 2-71 (Scheme 4.4).

Scheme 4.4. Synthesis of amides bearing bulky substituents on nitrogen.

Amine Product Yield

4-43 12%

4-44 —

4-45   7%

4-46 11%

Amine Product Yield

4-47 74%

4-48 39%

4-49 78%

4-50 90%

Other derivatives were found to be sensitive to the harsh acidicconditions used to deprotect the phenol in the penultimate step. Wherethis was the case, we used the following two-step Protocol (Scheme 4.5).Removal of the PMB protecting group was achieved in refluxing TFA andthe product was isolated by chromatography. Treatment with BBr₃ in DCMat room temperature revealed the phenol which was used withoutpurification. Oxidation under standard conditions with salcomineprovided the desired DNQ derivative. Derivatives 4-36 and 4-38 were notrecovered after precursors 4-59 and 4-61 were subjected to theoxidation; the reasons for this are unknown.

Scheme 4.5. Synthesis of DNQ derivatives bearing substituents sensitiveto HBr-mediated deprotection.

Com- Com- Yields over pound pound 2 steps 4-51

4-57 4-30 21% 4-52

4-58 4-36  0% 4-53

4-59 4-37 29% 4-54

4-60 4-38  0% 4-55

4-61 4-39 21% 4-56

4-62 4-41 40%

In addition to 4-36 and 4-38, derivatives 4-33, 4-40, and 4-42 were notsuccessfully synthesized. Unsurprisingly, the intramolecular amidationen route to t-butyl derivative 4-33 failed to provide the extremelycongested tricyclic product even under elevated temperature andprolonged heating. Under conditions for the global deprotection of 4-63or 4-64 en route to derivative 4-42 the pendant alcohol was converted tothe alkyl bromide and attempts to convert the bromide back to thealcohol were unsuccessful (Scheme 4.6). The reason that the conversionto the bromide during deprotection occurred during synthesis ofderivative 4-42 but not 4-41 is unknown.

NQO1-dependent cytotoxicity of a second set of DNQ derivatives. The DNQderivatives synthesized were assayed for their toxicity to MCF-7 cellswith or without DIC. The data from these experiments is organized inTable 4.2. Most of these compounds were found to be equipotent with DNQand showed a strong dependence on uninhibited NQO1. Linear alkyl chainsup to six carbons in length were tolerated (4-24), while the n-heptylderivative 4-25 was somewhat less active. The only branched alkylderivative that was significantly less toxic than DNQ was cyclooctylcompound 4-39. This tolerance of sterically-demanding substituentscorresponds with a flexible and promiscuous NQO1 active site. The pooractivity of hydroxyl derivative 4-41 may be a result of either poorbinding with the largely hydrophobic NQO1 active site or with anexcessively strong substrate-enzyme interaction because of hydrogenbonding. Alternatively, because the catalytic efficiency of 4-41 isequivalent to many of the more potent derivatives, 4-41 might be limitedby other factors such as cell permeability.

TABLE 4.2 Cytotoxicity and NQO1 dependence of second set of DNQderivatives. IC₅₀ vs. MCF-7 cells Fold (

 M ± s.e.) protection k_(cat)/K_(M) Compound +25 

 M DIC by DIC (M⁻¹s⁻¹× 10⁷) DNQ 0.13 ± 0.02 1.8 ± 0.3 14 6.7 4-22 0.27 ±0.04 1.5 ± 0.4 5.5 7.9 4-23 0.20 ± 0.01 2.0 ± 0.9 10 5.0 4-24 0.29 ±0.04 3.0 ± 0.6 10 5.8 4-25 0.42 ± 0.11 4.5 ± 0.4 11 — 4-26 0.77 ± 0.192.2 ± 0.6 2.8 3.6 4-27 6.3 ± 1.1 6.8 ± 1.6 1.1 0.63 4-28 36 ± 11 31 ± 100.9 0.22 4-29 3.8 ± 1.2 4.8 ± 1.1 1.3 0.46 4-30 0.24 ± 0.02 2.1 ± 0.88.6 2.0 4-31 0.23 ± 0.02 1.1 ± 0.1 4.8 6.1 4-32 0.18 ± 0.03 1.8 ± 0.5 107.3 4-34 0.23 ± 0.01 2.1 ± 0.7 8.9 4.9 4-35 0.22 ± 0.04 1.6 ± 0.6 7.16.7 4-37 0.22 ± 0.01 1.4 ± 0.2 6.1 2.8 4-39 0.83 ± 0.07 4.1 ± 0.8 3.81.7 4-41 0.68 ± 0.15 3.4 ± 0.6 5.0 3.9

Solubility of DNQ derivatives. To establish the relationship betweenstructure and solubility of DNQ derivatives we assessed the solubilityof every derivative in PBS buffer (pH 7.4), DCM, THF, DMSO, and amixture of 33% methanol in DCM. The results are organized in Table 4.3and shown graphically in the following sections. The descriptions ofFIGS. 7-11 note “active” compounds (IC₅₀<500 nM) and generally“inactive” compounds (IC₅₀>500 nM).

TABLE 4.3 Solubility of DNQ derivatives in PBS, HPβCD, and severalorganic solvents. 2:1 PBS HPβCD DMSO CH₂Cl₂ CH₂Cl₂:MeOH Compound (

 M) (mM) (mM) (mM) (mM) DNQ 115 3.3 3.1 0.7 8.5 4.8  439 4.4 31 5.5 374.9  59 — 10 6.1 18 4.10 <5 — <0.1 1.8 6.4 4.11 105 — 16 11 35 4.12 35 —4.2 2.0 10 4.13 <5 — <0.1 1.5 5.9 4.14 319 5.7 24 9.5 19 4.15 39 6.3 8.93.9 32 4.16 <5 — 1.2 34 34 4-22 17 4.0 10 7.6 30 4-23 <5 5.0 17 22 954-24 <5 6.0 18 32 140 4-25 <5 3.0 13 30 — 4-26 <5 — 14 24 75 4-27 <5 —10 17 49 4-28 <5 — 7.1 35 64 4-29 84 — 21 10 46 4-30 41 3.7 21 5.6 794-31 115 13   110 180 140 4-32 6 4.7 8.6 9.7 58 4-34 24 9.4 16 23 1004-35 7 19   15 31 120 4-37 114 4.3 25 13 51 4-39 24 — 71 120 46 4-41 485— 24 0.3 11

Aqueous solubility. The aqueous solubility of the compounds wasdetermined by an LC-MS assay as described in the specification above. Weapplied the calibration curve generated for DNQ to the derivatives underthe assumption that the UV profile of the derivatives of DNQ would besimilar to that of DNQ.

After sonication and filtration most of the aqueous solutions werecolorless. The solutions of DNQ, 4-31, and 4-37 were faintly yellowwhile 4-8, 4-14 and 4-41 were substantially more colored. The solutionof 4-8, 4-14 and 4-41 were diluted 10-fold with PBS prior to LC-MSanalysis to ensure that the concentrations fell within the range of thecalibration curve. The results are given in Table 4.3 and FIG. 7.

As predicted, compounds 4-8 and 4-14 were more soluble than DNQ despitetheir increased lipophilicity. As the lipophilicity of derivativesincreased further, however, solubility deceased. A few derivatives, suchas 4-31 and 4-37, were substantially more soluble than derivatives ofsimilar lipophilicity such as 4-22 and 4-23 indicating some advantage ofmoderate steric bulk near the nitrogen. It is unsurprising that compound4-41 was the most soluble DNQ derivative. This highlights the necessityof making a series of alcohol-bearing derivatives in an attempt to findmore active derivatives.

Solubility of DNQ derivatives in HPβCD. Although we were pleased to findactive derivatives of DNQ that were up to 4-fold more soluble inwater—sufficiently soluble for continuous IV infusion for humanadministration—this level of solubility was insufficient for animalstudies by bolus injection. We therefore determined the solubility ofall the most active derivatives (IC₅₀ below 500 nM) in 20% HPβCDsolution. We used the same phi modulation protocol outlined for DNQdescribed in the specification above. The results are organized in Table4.3 and FIG. 8. While all compounds were more soluble in HPβCD than inbuffer alone, the fold increase in solubility varied widely.

We observed that, in general, the more lipophilic the compound thegreater the enhancement of solubility by HPβCD. Of particular note is4-35 which, although very poorly soluble in water, is the most solublederivative in HPβCD, with a fold enhancement of ˜3000. This foldenhancement in solubility is among the highest ever reported for HPβCDformulations (Stella and He, “Cyclodextrins” Toxicol. Pathol. 2008, 36,30). In addition to being the most soluble derivative, 4-35 also appearsto be the least sensitive to acidic pH. As the pH of the solution islowered beyond the pKa of the N—H proton, the derivatives becomesubstantially less soluble and precipitate rapidly. This is especiallytrue for 4-8. However, once dissolved with HPβCD in basic solution, 4-35remained in solution even when the pH was rendered acidic. This mayresult from a binding orientation between 4-35 and HPβCD which favorsboth a tight binding of the t-butyl in the cyclodextrin pocket as wellas a strong hydrogen-bond between the deprotonated lactam and thehydroxyls at the rim of the cavity. This hydrogen bonding must besufficiently strong to “hide” the deprotonated lactam from protons inthe surrounding bulk water. This phenomenon of stability to acidic pH isdisplayed to varying lesser extents with the other derivatives. It islikely that compound 4-8 is oriented with the ethyl group in the cavityof the cyclodextrin which results in the deprotonated lactam beingexposed to the bulk solvent. This would explain the strong dependence ofthe solubility of this compound on the pH of the solution.

Solubility in organic solvents. The solubility of DNQ derivatives inorganic solvents was determined by measuring the amount of a giverssolvent required to dissolve a known amount of compound. Solvent wasadded to the compound in 50 μL aliquots and the slurry was sonicated.This process was repeated until all the solid had dissolved, leaving aclear red solution. The endpoint was generally very well defined.Solubility was assessed in DMSO, dichloromethane, and 33% methanol indichloromethane (FIGS. 9, 10, and 11, respectively).

All but a few derivatives were more soluble in organic solvents than wasDNQ. Only alcohol derivative 4-41 and the derivatives with dodecylchains (4-10 and 4-13) were less soluble. Two compounds, activederivative 4-31 and a less active derivative 4-39, stood out as far moresoluble than the others in both DMSO and dichloromethane. While weassume that the solubility of cyclooctyl compound 4-39 stems from itscomplete disruption of π-stacking as well as its high lipophilicity, wecannot explain the impressive solubility of 4-31. Other compounds, suchas active n-hexyl derivative 4-24 displayed good solubility and appearto be reasonable candidates for micellar formulations.

Properties of 4-31 measured by Absorption Systems. A sample of compound4-31 was delivered to Absorption Systems, a company that specializes inpreclinical formulation and stability studies. They examined a largenumber of formulation and excipients but did not find conditions whichbetter solubilized 4-31 than the HPβCD formulation described above. Theymeasured a few physical properties of 4-31, including pK_(a), logP andlogD. Through potentiometric titrations they measured the pK_(a) of 4-31to be 8.0. The logP (octanol/0.15 M KCl partition coefficient ofunionized compound) was found to be 1.8. The logD (octanol/0.15 M KClpartition coefficient of ionized compound) values at various pHs areshown in Table 4.4.

TABLE 4.4 logD of compound 4-31 between pH 3.0 and pH 9.0. pH 3.0 4.06.8 7.0 8.0 9.0 logD 1.78 1.78 1.76 1.75 1.49 0.76

Absorption Systems measured the stability of 4-31 during incubation inrat, dog, and human plasma, whole blood, and liver microsomes. Theyreport that 4-31 shows no sign of degradation after 2 hours in plasma orwhole blood or after 1 hour in liver microsomes from any of the threespecies. This is in strong contrast to β-lap, which has been reported tobe completely degraded within about 30 min in whole blood.

Maximum tolerated dose in mice. Thirteen of the 30 derivativessynthesized were found to be approximately equipotent to DNQ in cellculture and were at least as soluble as DNQ in HPβCD. All of thesederivatives are currently being assessed for their maximum tolerateddose (MTD) in healthy mice. Compounds are formulated in HPβCD anddelivered by IP injection to two mice once dally for five days. Initialresults for DNQ and nine of the derivatives are shown in Table 4.5. MTDis displayed both in mg/kg and in μmol/kg to facilitate directcomparison with DNQ without the larger molecular weights of thederivatives obfuscating the results. All the derivatives cause the samephenotype in mice: at the MTD, mice are lethargic and unresponsive totouch for up to one hour, after which time they recover fully. Thus far,4-35 is the best tolerated; it is tolerated at 3.5 folder higherconcentration than DNQ.

TABLE 4.5 Maximum tolerated dose of DNQ and nine derivatives in mice.mg/kg

 mol/kg DNQ 5 18 4-8  ≦10 ≦35 4-14 10 35 4-15 ≧14 ≧44 4-23 ≧15 ≧44 4-24≧16 ≧44 4-31 14 44 4-32 15 44 4-34 ≧15 ≧44 4-35 22 62

In summary, we have reported our efforts to develop deoxynyboquinonederivatives as new candidates for the personalized treatment of cancer.We identified DNQ through a high-throughput screen for cytotoxicity andsubsequently designed a flexible and modular synthetic route to DNQ andits derivatives. We demonstrated that DNQ kills cells through rapid ROSgeneration and that ROS generation occurred through a 2-electronbioreduction/oxidation process mediated exclusively by NQO1. We showedthat DNQ was able to slow tumor growth in a mouse model of cancer butthat the most efficacious doses were poorly tolerated by mice. We thensynthesized a library of derivatives of DNQ and assessed them forcytotoxicity, solubility, and tolerance in mice. Through this process weidentified a number of compounds that are more soluble than, equipotentto, and better tolerated by mice than DNQ. The most promising of thesederivatives are being assessed in mouse models of cancer to identify acandidate molecule for further evaluation in preclinical and humanclinical trials.

Preparation of DNQ Derivatives. The materials and methods are analogousto those described for Example 1.

General protocol A: Amidation of ester. To a solution of alkynyl ester(1 equiv.) in methanol (2 mM), chilled in an ice-water bath was addedalkyl amine (1.2 equiv.). The reaction was stirred at 0° C. for 14 h.The solvent was evaporated directly from the flask and the residue wasseparated by silica gel chromatography to yield the desired alkynylamide.

General protocol B: Amidation of acid chloride. To an oven-dried Schlenkflask with a stir bar was added iodoacid 2-73 and the flask wasevacuated and backfilled with argon. Dry CH₂Cl₂ was added and thesolution was chilled on an ice-water bath. Oxalyl chloride (3 equiv.)was added by syringe and the cold bath was removed. After 5 h at roomtemperature the volatile components were evaporated directly from theflask. Dry CH₂Cl₂ (10 mL) was added to the residual oil and the vial waschilled on a dry ice/isopropanol bath. Freshly distilled p-methoxybenzylamine (1.1 equiv.) was added dropwise by syringe followed by NEt₃ (1.2equiv.). The mixture was stirred for 10 minutes then was allowed to warmto RT. 1 M HCl (20 mL) was added and the solution was poured info aseparatory funnel with CH₂Cl₂ (10 mL), shaken and separated. The aqueousfraction was extracted with CH₂Cl₂ (4×10 mL) then dried over MgSO₄ andevaporated. The residue was purified by silica gel chromatography.

General protocol C: Hydroiodination. Alkynyl amide (1 equiv.), NaI (2equiv.), and acetic acid (10 equiv.) were combined and heated to 115° C.for 8 h. Reaction completion was determined by removing aliquots for¹H-NMR analysis. The deep red reaction mixture was diluted with waterand CH₂Cl₂, treated with NaHSO until colorless, and carefullyneutralized with a saturated aqueous solution of NaHCO₃. This mixturewas poured info a separatory funnel with CH₂Cl₂, shaken and separated.The aqueous fraction was extracted with CH₂Cl₂. The combined organicfractions were washed with brine, dried over MgSO₄, and evaporated toyield the desired iodoamide.

General protocol D: Suzuki cross-coupling. To a Schlenk flask with astir bar was added pure (recrystallized) bispinacolboronate 4-6 (1equiv.), PdCl₂(dppf) (20 mol %), K₂CO₅ (6 equiv.), and both desirediodoamides (1.3 equiv, of amide bearing PMB, 1.5 equiv. of N-alkylamide) and the flask was evacuated and backfilled with argon threetimes. Water (1 mL) and DME (9 mL) were added by syringe after degassingthe solvents by bubbling with argon for 45 minutes. The flask wasplunged into an oil bath at 80° C. for 3 h. The mixture was poured intoa separatory funnel and diluted with water (5 mL). The mixture wasextracted with EtOAc (2×mL). The combined organic extracts were driedover MgSO₄, filtered and evaporated to a deep red oil. The crude productwas dissolved in CH₂Cl₂ and separated by silica gel chromatography. Thepurity of the diamide product was highly variable and the product wassubjected to intramolecular amidation without further purification.

General protocol E: intramolecular aryl amidation. In a Schlenk flask ora vial with a Teflon-lined cap were combined the diamide startingmaterial, K₂CO₃ (6 equiv.), Pd/X-Phos (10 mol %), and X-Phos (10 mol %).The flask was cycled between vacuum and argon three times andargon-sparged i-PrOH was added by syringe. The mixture was heated to 80°C. with stirring for 14 h. Insoluble materials were removed byfiltration through Celite and rinsed with CH₂Cl₂. The filtrate wasevaporated and the residue was used directly in the next step.

General protocol F: HBr deprotection. The crude diazaanthracene wasdissolved in 48% HBr and heated to 110° C. After 19 hours the reactionwas removed from heat. The mixture was cooled on an ice bath and wascarefully rendered basic by adding 10 M NaOH. The residual solid wasremoved by filtration through hardened filter paper and discarded. Thefiltrate was rendered acidic with 1 M HCl, whereupon a colloidalprecipitate formed. The mixture was then centrifuged (3220×g for 5minutes). The resulting semi-compact gelatinous solid was collected byfiltration through hardened filter paper and dried to a constant massunder vacuum to yield the desired diazaanthracenol in frequently highpurity as assessed by NMR.

General protocol G: BBr₃ deprotection and oxidation without isolation.For substrates that proved sensitive to global deprotection by HBr, thefollowing protocol was employed. The product of intramolecular amidation(General Protocol E) was dissolved in TFA and heated to reflux for 1 h.The solvent was then evaporated and the residue was purified by silicagel chromatography.

In a Schlenk flask containing the PMB-deprotected material under Ar wasadded DCM and the solution was cooled in a dry ice/isopropanol bath.BBr₃ (6 equiv.) was added by syringe and the solution was stirred untilstarting material was consumed as shown by TLC. Residual BBr₃ wasquenched by the addition of conc. NaHCO₃ solution until pH neutral. Thesolvents were evaporated. The residue was oxidized and the resulting DNQderivative was purified using General Protocol H.

General protocol H: Oxidation. To a flask containing thediazaanthracenol starting material was added salcomine (10 mol %) andDMF. A balloon containing O₂ was fitted over the mouth of the flask andthe slurry was stirred at room temperature. The solid dissolved afterabout 30 minutes. After 3 h stirring, the mixture was diluted with onevolume each of DCM and hexanes and loaded directly onto a chromatographycolumn consisting of a layer of basic alumina (5 cm) under a layer ofsilica gel (5 cm) prepared in DCM. The column was flushed withincreasing amounts of methanol (0-2%) in DCM until the red product bandentered the alumina layer which retained the product, allowing coelutingimpurities to be removed. The product was then released from the basicalumina by adding 1% HOAc to the mobile phase. The red fractions wereevaporated and purified by chromatography through silica gel (0-5% MeOHin DCM) to yield the desired DNQ derivative as an orange, red, orred-pink solid.

Synthesized by General Protocol A. 78% yield.

¹H-NMR (CDCl₃, 500 MHz): δ 6.1 (bs, 1H), 2.99 (d, 3H, J=5.0 Hz, minorrotamer), 2.81 (d, 3H, J=5.0 Hz, major rotamer), 2.35 (t, 2H, J=7.0 Hz,minor rotamer), 2.22 (t, 2H, J=7.0 Hz, major rotamer), 1.19 (t, 3H,J=7.5 Hz, minor rotamer), 1.13 (t, 3H, J=7.5 Hz, major rotamer). ¹³C-NMR(CDCl₃, 125 MHz): δ 154.50, 88.44, 74.95, 26.58, 12.93, 12.40.

Synthesized by General Protocol C. 92% yield.

¹H-NMR (CDCl₃, 500 MHz): δ 6.28 (q, 1H, J=1.5 Hz, vinyl CH), 5.9 (bs,1H, NH), 2.88 (d, 3H, J=5.0 Hz, NCH₃), 2.62 (dq, 2H, J=1.5 Hz, 7.5 Hz,allylic CH₂), 1.11 (t, 3H, J=7.5 Hz, —CH₃). ¹³C-NMR (CDCl₃125 MHz): δ166.14, 127.91, 115.56, 40.94, 26.39, 14.67. HRMS (ESI-TOF) calcd forC₆H₁₁NOI (M+H)⁺: 239.9885. found: 239.9885.

Synthesized by General Protocols D, E, F, and H. 7.4% yield over 4steps.

¹H-NMR (2:1 CDCl₃:CD₃OD, 500 MHz): δ 6.81 (d, 1H, J=1.0 Hz, vinyl CH),6.67 (d, 1H, J=1.0 Hz, vinyl CH), 3.92 (s, 3H), 3.09 (dq, 2H, J=7.0, 0.5Hz, allylic CH₂), 2.64 (d, 3H, J=1.5 Hz, allylic CH₃), 1.26 (t, 3H,J=7.5 Hz, CH3). ¹³C-NMR (CDCl₃, 125 MHz): δ 181.33, 175.31, 162.29,161.90, 155.57, 151.68, 140.24, 138.50, 127.15, 125.27, 118.83, 115.06,33.98, 27.88, 21.83, 13.55. HRMS (ESI-TOF) calcd for C₁₆H₁₅N₂O₄ (M+H)⁺:299.1032, found: 299.1034.

Synthesized by General Protocol A. 92% yield.

¹H-NMR (CDCl₃, 500 MHz): δ 6.04 (bs, 1H, major rotamer NH), 5.89 (bs,1H, minor rotamer), 2.99 (d, 3H, J=5.0 Hz, minor rotamer), 2.81 (d, 3H,J=5.0 Hz, major rotamer), 2.33 (t, 2H, J=7.0 Hz, minor rotamer), 2.22(t, 2H, J=7.0 Hz, major rotamer), 1.59 (sext, 2H, J=7.0 Hz, minorrotamer), 1.54 (sext, 2H, J=7.0 Hz, major rotamer), 0.99 (t, 3H, J=7.5Hz, minor rotamer), 0.96 (t, 3H, J=7.5 Hz, major rotamer). ¹³C-NMR(CDCl₃, 125 MHz); δ 54.47 (major), 87.18 (major), 75.76 (major), 29.87(minor), 26.58 (major), 21.43 (major), 20.97 (minor), 20.65 (major),13.60 (major). HRMS (ESI) calcd for C₇H₁₂NO (M+H)⁺: 126.0919, found:126.0920.

Synthesized by General Protocol C. 96% yield.

¹H-NMR (CDCl₃, 500 MHz): δ 6.40 (bs, 1H, NH), 6.30 (s, 1H, vinyl CH),2.82 (d, 3H, J=5.0 Hz, NCH₃), 2.53 (t, 2H, J=7.0 Hz, allylic CH₂), 1.54(sext, 2H, J=7.5 Hz), 0.86 (t, 3H, J=7.5 Hz), ¹³C-NMR (CDCl₃, 125 MHz):δ 165.86, 128.49, 114.01, 49.03, 26.30, 22.52, 12.82. HRMS (ESI-TOF)calcd for C₇H₁₃NOI (M+H)⁺: 254.0042, found: 254.0045.

Synthesized by General Protocols D, E, F, and H, 3.4% yield over 4steps.

¹H-NMR (CDCl₃, 500 MHz): δ 6.80 (s, 1H, vinyl CH), 6.68 (d, 1H, J=1.0Hz, vinyl CH), 3.93 (s, 3H), 2.98 (t, 2H, J=7.5 Hz, allylic CH₂), 2.62(d, 3H, J=1.0 Hz, allylic CH₃), 1.61 (q, 2H, J=7.5 Hz), 1.03 (t, 3H,J=7.5 Hz, CH(CH₃)₂). HRMS (ESI-TOF) calcd for C₁₇H₁₇N₂O₄ (M+H)⁺:313.1188, found: 313.1189.

To an oven-dried Schlenk flask was added 1-tetradecyne (0.748 g, 3.85mmol) and THF (10 mL). Chilled to −78° C. Added n-BuLi (2.7 mL, 4.32mmol) dropwise then stirred for 10 minutes. Added ethyl chloroformate(0.56 mL, 5.86 mmol) then allowed the reaction to warm to RT. Thesolvent was evaporated and the residue was purified by silica gelchromatography. Product was collected as a colorless oil (1.01 g, 3.79mmol, 98.5% yield).

¹H-NMR (CDCl₃, 500 MHz): δ 4.19 (q, 2H, J=7.5 Hz), 2.30 (t, 2H, J=7.5Hz), 1.56 (pent, 2H, J=7.5 Hz), 1.37 (bpent, 2H, J=8.0 Hz), 1.29 (t, 3H,J=7.5 Hz), 1.28-1.21 (m, 16H), 0.86 (t, 3H, J=7.0 Hz). ¹³C-NMR (CDCl₃,125 MHz): δ 154.06, 89.66, 73.33, 61.91, 32.10, 29.82, 29.81, 29.77,29.60, 29.53, 29.21, 29.04, 27.73, 22.87, 14.29, 14.21.

Synthesized by General Protocol A. 66% yield.

¹H-NMR (CDCl₃, 500 MHz): δ 6.42 (bs, 1H, major rotamer NH), 6.24 (bs,1H, minor rotamer NH), 2.93 (d, 3H, J=5.0 Hz, minor rotamer NCH₃), 2.75(d, 3H, J=5.0 Hz, major rotamer NCH₃), 2.29 (t, 2H, J=7.0 Hz, minorrotamer allylic CH₂), 2.18 (t, 2H, J=7.0 Hz, J=7.0 Hz, major rotamerallylic CH₂), 1.50 (pent, 2H, J=7.0 Hz, minor rotamer), 1.45 (pent, 2H,J=7.5 Hz, major rotamer), 1.29 (bpent, 2H, J=7.5 Hz, major rotamer),1.25-1.13 (m, 16H), 0.79 (t, 3H, J=7.0 Hz). ¹³C-NMR (CDCl₃, 125 MHz): δ157.35 (minor), 154.47 (major), 94.59 (minor), 87.17 (major), 75.50(major), 73.14 (minor), 31.92 (major), 29.66 (major), 29.64 (2C, major),29.48 (major), 29.36 (major), 29.10 (major), 28.90 (major), 27.83(major), 26.43 (major), 22.69 (major), 18.57 (major), 14.11 (major).

Synthesized by General Protocol C. 100% yield.

¹H-NMR (CDCl₃, 500 MHz): δ 6.27 (s, 1H, vinyl CH), 5.74 (bs, 1H, NH),2.89 (d, 3H, J=4.5 Hz), 2.59 (t, 2H, J=7.5 Hz), 1.56 (bt, 2H, J=7.0),1.33-1.22 (m, 18H), 0.88 (t, 3H, J=7.0 Hz). ¹³C-NMR (CDCl₃, 125 MHz): δ165.96, 128.50, 114.45, 47.23, 32.07, 29.81, 29.80, 29.77, 29.66, 29.51,29.50, 29.36, 28.44, 26.36, 22.84, 14.29.

Synthesized by General Protocols D, E, F, and H. 7.0% yield over 4steps.

¹H-NMR (2:1 CDCl₃:CD₃OD, 500 MHz): δ 6.78 (s, vinyl CH), 6.67 (d, 1H,J=1.0 Hz, vinyl CH), 3.91 (s, 3H), 3.03 (t, J=8.0 Hz), 2.64 (d, 3H,J=1.0 Hz, allylic CH₃), 1.58 (p, 2H, J=7.5 Hz), 1.44 (p, 2H, J=7.5 Hz),1.27-1.4 (m, 16H), 0.89 (t, 3H, J=7.5 Hz). ¹³C-NMR (d-TFA, 125 MHz): δ182.13, 176.16, 166.58 (bs), 163.92 (bs), 160.66, 141.89, 139.75, 128.19(bs), 126.92, 125.93, 120.93, 38.25, 37.45, 33.96, 32.05, 31.63 (2C),31.57, 31.52, 31.41, 31.36, 31.15, 24.53, 23.41, 14.85.

Synthesized by General Protocols D, E, F, and H. 6.2% yield over 4steps.

¹H-NMR (2:1CDC₃:CD₃OD, 500 MHz): δ 6.78 (d, 1H, J=1.0 Hz, vinyl CH),6.70 (s, 1H, vinyl CH), 3.92 (s, 3H), 3.09 (qd, 2H, J=7.5, 1.0 Hz), 2.64(d, 3H, J=1.0 Hz, allylic CH₃), 126 (t, 3H, J=7.5 Hz, CH(CH₃)₂).

Synthesized by General Protocols D, E, F, and H. 11% yield over 4 steps.

¹H-NMR (CDCl₃, 500 MHz): δ 9.48 (bs, 1H, NH), 6.79 (d, 1H, J=1.5 Hz,vinyl CH), 6.69 (s, 1H, vinyl CH), 3.93 (s, 3H), 3.00 (t, 2H, J=7.5 Hz)2.61 (d, 3H, J=1.0 Hz, allylic CH₃), 1.62 (sext, 2H, J=7.5 Hz), 1.04 (t,3H, J=7.5 Hz). HRMS (ESI-TOF) calcd for C₁₇H₁₇N₂O₄ (M+H)⁺: 313.1188,found: 313.1187.

Synthesized by General Protocols D, E, F, and H. 20% yield over 4 steps.

¹H-NMR (2:1 CDCl₃:CD₃OD, 500 MHz): δ 6.78 (s, vinyl CH), 6.67 (s, 1H),3.92 (s, 3H), 3.03 (t, J=7.5 Hz), 2.64 (d, 3H, J=1.0 Hz, allylic CH₃),1.58 (p, 2H, J=7.5 Hz), 1.44 (p, 2H, J=7.5 Hz), 1.27-1.4 (m, 16H), 0.89(t, 3H, J=7.5 Hz), ¹³C-NMR (d-TFA, 125 MHz): δ 182.21, 176.18, 166.64,166.55, 165.56, 158.87, 141.347, 140.27, 128.14, 127.13, 126.09, 120.54,37.93, 36.73, 33.95, 31.84, 31.61 (2H), 31.54, 31.42, 31.38, 31.34,31.13, 24.50 24.37, 14.79.

Synthesized by General Protocols D, E, F, and H. 17% yield over 4 steps.

¹H-NMR (CDCl₃, 500 MHz): δ 10.28 (bs, 1H), 6.83 (s, 1H, vinyl CH), 6.75(s, 1H, vinyl CH), 3.93 (s, 3H), 3.07 (dq, 2H, J=7.5 Hz, 1.0 Hz), 3.04(dq, 2H, J=7.5 Hz, 1.0 Hz), 1.25 (t, 3H, J=7.5 Hz), 1.24 (t, 3H, J=7.5Hz). ¹³C-NMR (2:1 CDCl₃:CD₃OD, 125 MHz): δ 181.29, 175.34, 162.29,162.15, 157.28, 155.52, 140.00, 138.78, 33.96, 27.90, 27.27, 13.62,13.34.

Synthesized by General Protocol A. 68% yield.

¹³C-NMR (CDCl₃, 125 MHz): δ 153.76 (major), 89.96 (major), 75.94(major), 43.21 (minor), 39.66 (major), 32.77 (minor), 31.51 (major),21.43 (major), 20.96 (minor), 20.67 (major), 20.14 (major), 19.83(minor), 13.8 (major), 13.8 (minor), 13.6 (major), 13.6 (minor). HRMS(ESI) calcd for C₁₀H₁₅NO (M+H)⁺: 168.1388, found: 168.1382.

Synthesized by General Protocol C. 98% yield.

¹H-NMR (CDCl₃, 500 MHz): δ 6.26 (s, 1H, vinyl CH), 3.25 (q, 2H, J=7.0Hz), 2.51 (t, 2H, J=7.5 Hz), 1.54 (sext, 2H, J=7.5 Hz), 1.48 (pent, 2H,J=7.5 Hz), 1.32 (sext, 2H, J=8.0 Hz), 0.86 (t, 3H, J=7.0 Hz), 0.85 (t,3H, J=7.5 Hz). ¹³C-NMR (CDCl₃, 125 MHz): δ 165.13, 128.87, 113.51,49.01, 39.36, 31.54, 22.51, 20.27, 13.81, 12.83, HRMS (ESI-TOF) calcdfor C₁₀H₁₉NOI (M+H)⁺: 296.0511, found: 296,0503.

Synthesized by General Protocols D, E, F, and H. 6.3% yield over 4steps.

¹H-NMR (CDCl₃, 500 MHz): δ 9.46 (s, 1H, NH), 6.77 (s, 1H, vinyl CH), 6.6(d, 1H, J=1.0 Hz, vinyl CH), 4.50 (m, 2H), 2.97 (t, 2H, J=7.5 Hz,allylic CH₂), 2.61 (d, 3H, J=1.0 Hz, allylic CH₃), 1.69 (pent, 2H, J=8.0Hz), 1.60 (sext, 2H, J=7.5 Hz), 1.47 (sext, 2H, J=8.0 Hz), 1.03 (t, 3H,J=7.5 Hz, CH₃), 0.99 (t, 3H, J=7.5 Hz, CH₃). ¹³C-NMR (CDCl₃, 125 MHz): δ181.73, 175.37, 161.47, 160.91, 153.23, 151.31, 139.49, 137.78, 128.40,127.82, 119.67, 114.98, 46.35, 39.15, 31.40, 23.14, 22.40, 20.40, 14.28,13.93. HRMS (ESI-TOF) calcd for C₂₀H₂₃N₂O₄ (M+H)⁺: 355.1658, found:355.1655.

Synthesized by General Protocols D, E, F, and H. 6.7% yield over 4steps.

¹H-NMR (CDCl₃, 500 MHz): δ 9.52 (s, 1H, NH), 6.77 (d, 1H, J=1.0 Hz,vinyl CH), 6.68 (s, 1H, vinyl CH), 4.52-4.49 (m, 2H), 2.99 (t, 2H, J=7.5Hz, allylic CH₂), 2.59 (d, 3H, J=1.0 Hz, allylic CH₃), 1.68 (pent, 2H,J=7.5 Hz), 1.61 (sext, 2H, J=8.0 Hz, CH₂CH₂CH₃), 1.47 (sext, 2H, J=8.0Hz, CH₂CH₂CH₃), 1.04 (t, 3H, J=7.5 Hz, CH₃), 1.00 (t, 3H, J=7.5 Hz,CH₃). ¹³C-NMR (CDCl₃, 125 MHz): δ 181.67, 175.28, 161.28, 160.96,155.41, 149.27, 138.84, 138.25, 128.69, 127.59, 120.12, 114.40, 46.20,36.30, 31.40, 23.53, 22.88, 20.39, 14.13, 13.92, HRMS (ESI-TOF) calcdfor C₂₀H₂₃N₂O₄ (M+H)⁺: 355.1658, found: 355.1658.

Synthesized by General Protocols D, E, F, and H. 13% yield over 4 steps.

¹H-NMR (CDCl₃, 500 MHz): δ 9.67 (s, 1H, NH), 6.77 (s, 1H, vinyl CH),6.68 (s, 1H, vinyl CH), 4.49 (m, 2H), 2.98 (t, 2H, J=7.0 Hz, allylicCH₂), 2.95 (t, 2H, J=7.0 Hz, allylic CH₃), 1.69 (pent, 2H, J=8.5 Hz,CH), 1.72-1.65 (m, 2H), 1.65-1.55 (m, 4H), 1.47 (sext, 2H, J=7.5 Hz),1.03 (t, 3H, J=7.0 Hz), 1.03 (t, 3H, J=7.5 Hz), 1.00 (t, 3H, J=7.5 Hz,CH₃). ¹³C-NMR (CDCl₃, 125 MHz): δ 181.70, 175.33, 161.47, 160.79,155.34, 153.19, 139.15, 138.01, 127.93, 127.62, 120.10, 114.69, 46.31,37.09, 36.23, 31.41, 23.25, 22.98, 20.40, 14.28, 14.14, 13.93. HRMS(ESI-TOF) calcd for C₂₂H₂₇N₂O₄ (M+H)⁺: 383.1971, found: 383.1969.

Synthesized by General Protocol A. 83% yield.

¹H-NMR (CDCl₃, 500 MHz): δ 6.25 (bs, 1H), 3.35 (pent, 2H, J=7.0 Hz,minor rotamer NCH₂), 3.23 (pent, 2H, J=7.5 Hz, major rotamer), 1.95 (s,3H, minor rotamer), 1.85 (s, 3H, major rotamer), 1.12 (t, 3H, J=7.5 Hz,minor rotamer), 1.08 (t, 3H, J=7.0 Hz, major rotamer). ¹³C-NMR (CDCl₃,125 MHz): δ 153.58 (major), 82.80 (major), 75.05 (major), 38.20 (minor),34.66. (major), 15.91 (minor), 14.51 (major), 3.93 (minor), 3.59(major). HRMS (ESI) calcd for C₆H₁₀NO (M+H)⁺: 112.0762, found: 112.0764.

Synthesized by General Protocol C. 98% yield.

¹H-NMR (CDCl₃, 500 MHz): δ 6.28 (bs, 1H, NH), 6.22 (s, 1H, vinyl CH),3.30 (pent, 2H, J=7.5 Hz, NCH₂), 2.59 (s, 3H, allylic CH₃), 1.12 (s,3H). ¹³C-NMR (CDCl₃, 125 MHz): δ 164.85, 129.28, 105.58, 35.75, 34.45,14.74. HRMS (ESI-TOF) calcd for C₆H₁₁NOI (M+H)⁺: 139.9885, found:139.9884.

Synthesized by General Protocols D, E, F, and H. 13% yield over 4 steps.

¹H-NMR (2:1 CDCl₃:CD₃OD, 500 MHz): δ 6.76 (d, 1H, J=1.0 Hz, vinyl CH),6.67 (d, 1H, J=1.0 Hz, vinyl CH), 4.51 (q, 2H, J=8.0 Hz), 2.36 (m, 6H,allylic CH₃), 1.45 (t, 3H, J=7.0 Hz).

Synthesized by General Protocol A. 71% yield.

¹H-NMR (CDCl₃, 500 MHz): δ 5.99 (bs, 1H), 3.31 (q, 2H, J=7.0 Hz, minorrotamer NCH₂), 3.20 (q, 2H, J=7.5 Hz, major rotamer NCH₂), 1.98 (s, 3H,minor rotamer allylic CH₃), 1.89 (s, 3H, major rotamer allylic CH₃),1.50 (sext, 2H, J=7.5 Hz), 0.89 (t, 3H, J=7.5 Hz). ¹³C-NMR (CDCl₃, 125MHz): δ 153.71 (major), 89.96 (major), 75.13 (major), 45.15 (minor),41.57 (major), 23.92 (minor), 22.69 (major), 11.39 (major), 11.18(minor), 3.69 (major).

Synthesized by General Protocol C. 98% yield.

¹H-NMR (CDCl₃, 500 MHz): δ 6.23 (d, 1H, J=1.5 Hz, vinyl CH), 6.22 (bs,1H, NH), 3.23 (d, 2H, J=7.0 Hz, NCH₂), 2.60 (d, 3H, J=1.5 Hz, allylicCH₃), 1.53 (sext, 2H, J=7.0 Hz), 0.90 (t, 3H, 7.5 Hz). ¹³C-NMR (CDCl₃,125 MHz): δ 164.98, 129.47, 105.47, 41.34, 35.76, 22.77, 11.61. HRMS(ESI-TOF) calcd for C₇H₁₃NOI (M+H)⁺: 254.0042, found: 254.0044.

Synthesized by General Protocols D, E, F, and H. 13% yield over 4 steps.

¹H-NMR (CDCl₃, 500 MHz): δ 6.77 (d, 1H, J=1.0 Hz, vinyl CH), 6.68 (d,1H, J=1.5 Hz, vinyl CH), 4.60 (m, 2H), 2.62 (d, 3H, J=1.5 Hz, allylicCH₃), 2.60 (d, 3H, J=1.0 Hz, allylic CH₃), 1.73 (sext, 2H, J=8.0 Hz,CH), 1.04 (t, 3H, J=7.5 Hz). ¹³C-NMR (CDCl₃, 125 MHz); δ 181.48, 175.09,161.87, 161.75, 151.69, 149.97, 139.36, 138.58, 127.43, 127.21, 119.48,114.79, 22.87, 22.20, 21.81, 10.72.

Synthesized by General Protocol A. 78% yield.

¹H-NMR (CDCl₃, 500 MHz): δ 6.01 (bs, 1H), 3.34 (q, 2H, J=7.0 Hz, minorrotamer NCH₂), 3.23 (q, 2H, J=6.0 Hz, major rotamer NCH₂), 1.98 (s, 3H,minor rotamer allylic CH₃), 1.89 (s, 3H, major rotamer allylic CH₃),1.46 (pent, 2H, J=7.0 Hz), 1.31 (sext, 2H, J=7.5 Hz), 0.88 (t, 3H, J=7.5Hz). ¹C-NMR (CDCl₃, 125 MHz); δ 153.71 (major), 89.96 (major), 75.13(major), 45.15 (minor), 41.57 (major), 23.92 (minor), 22.69 (major),11.39 (major), 11.18 (minor), 3.69 (major).

Synthesized by General Protocol C. 97% yield.

¹H-NMR (CDCl₃, 500 MHz): δ 6.23 (d, 1H, J=1.5 Hz, vinyl CH), 3.26 (q,2H, J=7.0 Hz, NCH₂), 2.59 (d, 3H, J=1.5 Hz, allylic CH₃), 1.48 (p, 2H,J=7.5 Hz), 1.32 (sext, 2H, J=7.5 Hz), 0.87 (t, 3H, J=7.5 Hz). ¹³C-NMR(CDCl₃, 125 MHz): δ 164.93, 129.47, 105.41, 39.35, 35.75, 31.55, 20.26,13.83. HRMS (ESI-TOF) calcd for C₈H₁₅NOI (M+H)⁺: 268.0198, found:268.0197.

Synthesized by General Protocols D, E, F, and H. 9.9% yield over 4steps.

¹H-NMR (CDCl₃, 500 MHz): δ 9.54 (bs, 1H), 6.77 (q, 1H, J=1.0 Hz, vinylCH), 6.68 (q, 1H, J=1.0 Hz, vinyl CH), 4.53-4.50 (m, 2H), 2.62 (d, 3H,J=1.0 Hz, allylic CH₃), 2.60 (d, 3H, J=1.0 Hz, allylic CH₃), 1.68 (pent,2H, J=7.5 Hz), 1.47 (sext, 2H, J=7.5 Hz), 1.00 (t, 3H, J=7.5 Hz). HRMS(ESI-TOF) calcd for C₁₇H₁₇N₂O₄ (M+H)⁺: 313.1188, found: 313.1190.

Synthesized by General Protocol A. 86% yield.

HRMS (ESI) calcd for C₉H₁₆NO (M+H)⁺: 154.1232, found: 154.1231.

Synthesized by General Protocol C. 81% yield.

¹H-NMR (CDCl₃, 500 MHz): δ 6.22 (d, 1H, J=1.5 Hz, vinyl CH), 5.77 (bs,1H, NH), 3.32 (q, 2H, J=7.0 Hz, NCH₂), 2.64 (d, 3H, J=1.5 Hz, allylicCH₃), 1.55 (pent, 2H, J=7.0 Hz), 1.35-1.30 (m, 4H), 0.89 (t, 3H, J=7.0Hz). HRMS (ESI-TOF) calcd for C₉H₁₇NOI (M+H)⁺: 282.0355, found:282.0356.

Synthesized by General Protocols D, E, F, and H. 11% yield over 4 steps.

¹H-NMR (2:1 CDCl₃:CD₃OD, 500 MHz): δ 6.76 (d, 1H, J=1.0 Hz, vinyl CH),6.67 (d, 1H, J=1.5 Hz, vinyl CH), 4.48-4.43 (m, 2H), 2.63 (d, 3H, J=1.5Hz, allylic CH₃), 2.63 (d, 3H, J=1.0 Hz, allylic CH₃), 1.77 (pent, 2H,J=7.5 Hz), 1.48-1.40 (m, 4H), 0.95 (t, 3H, J=7.0 Hz). ¹³C-NMR (2:1CDCl₃:CD₃OD, 125 MHz): δ 181.47, 175.05, 161.83, 161.68, 151.66, 149.89,139.31, 138.53, 127.40, 127.19, 119.45, 114.75, 46.52, 28.82, 28.48,22.87, 22.07, 21.81, 13.58. HRMS (ESI-TOF) calcd for C₁₉H₂₁N₂O₄ (M+H)⁺:341.1501, found: 341.1496.

Synthesized by General Protocol A. 79% yield.

¹H-NMR (CDCl₃, 500 MHz): δ 5.85 (bs, 1H, major rotamer NH), 3.35 (q, 2H,J=7.0 Hz, minor rotamer), 3.23 (q, 2H, J=7.0 Hz, major rotamer), 2.00(s, 3H, minor rotamer), 1.91 (s, 3H, major rotamer), 1.48 (pent, 2H,J=7.0 Hz), 1.33-1.23 (m, 6H), 0.86 (t, 3H, J=7.0 Hz). HRMS (ESI) calcdfor C₁₀H₁₈NO (M+H)⁺: 168.1388, found: 168.1391.

Synthesized by General Protocol C. 98% yield.

¹H-NMR (CDCl₃, 500 MHz): δ 6.22 (q, 1H, J=1.5 Hz, vinyl CH), 5.73 (bs,1H, NH), 3.32 (q, 2H, J=7.0 Hz), 2.64 (d, 3H, J=1.5 Hz, allylic CH₃),1.55 (pent, 2H, J=7.0 Hz), 1.38-1.27 (m, 6H), 0.88 (t, 3H, J=7.0 Hz).HRMS (ESI-TOF) calcd for C₁₀H₁₉NOI (M+H)⁺: 296.0511, found: 296.0510.

Synthesized by General Protocols D, E, F, and H. 12% yield over 4 steps.

¹H-NMR (2:1 CDCl₃:CD₃OD, 500 MHz): δ 6.76 (s, 1H), 6.67 (s, 1H), 4.45(m, 2H), 2.63 (d, 3H, J=1.0 Hz, allylic CH₃), 2.63 (d, 3H, J=1.0 Hz,allylic CH₃), 1.76 (pent, 2H, J=7.5 Hz), 1.46 (pent, 2H, J=7.0 Hz),1.40-1.34 (m, 4H), 0.92 (t, 3H, J=7.0 Hz). ¹³C-NMR (2:1 CDCl₃:CD₃OD, 125MHz): δ 181.46, 175.04, 161.82, 161.66, 151.65, 149.88, 139.30, 138.53,127.40, 127.20, 119.44, 114.74, 46.57, 31.18, 28.76, 26.36, 22.87,22.35, 21.81, 13.58. HRMS (ESI-TOF) calcd for C₂₀H₂₃N₂O₅ (M+H)⁺:355.1658, found: 355.1660.

Synthesized by General Protocol A. 55% yield.

¹H-NMR (CDCl₃, 500 MHz): δ 5.95 (bs, 1H, NH), 3.34 (q, 2H, J=7.0 Hz,minor rotamer NCH₂), 3.22 (dt, 2H, J=7.0 Hz, major rotamer), 1.98 (s,3H, minor rotamer), 1.89 (s, 3H, major rotamer), 1.47 (pent, 2H, J=7.5Hz), 1.30-1.20 (m, 8H), 0.84 (t, 3H, J=7.0 Hz). ¹³C-NMR (CDCl₃, 125MHz): δ 156.51 (minor), 153.65 (major), 89.75 (minor), 82.97 (major),75.14 (major), 72.80 (minor), 43.44 (minor), 39.93, (major), 31.84(major), 30.67 (minor), 29.44 (major), 29.04 (major), 28.97 (minor),26.94 (major), 26.59 (minor), 22.69 (major).

Synthesized by General Protocol C. 86% yield.

¹H-NMR (CDCl₃, 500 MHz): δ 6.24 (q, 1H, J=1.5 Hz, vinyl CH), 5.73 (bs,1H, NH), 3.33 (q, 2H, J=6.5 Hz), 2.65 (d, 3H, J=1.5 Hz, allylic CH₃),1.55 (pent, 2H, J=7.0 Hz), 1.38-1.23 (m, 8H), 0.88 (t, 3H, J=7.0 Hz).

Synthesized by General Protocols D, E, F, and H. 10(5% yield over 4steps.

¹H-NMR (2:1 CDCl₃:CD₃OD, 500 MHz): δ 6.76 (q, 1H, J=1.5 Hz, vinyl CH),6.66 (q, 1H, J=1.0 Hz, vinyl CH), 4.47-4.44 (m, 2H), 2.63 (d, 3H, J=1.0Hz, allylic CH₃), 2.63 (d, 3H, J=1.0 Hz, allylic CH₃), 1.77 (bpent, 2H,J=8.0 Hz), 1.46 (bpent, 2H, J=8.0 Hz), 1.42-1.26 (m, 6H), 0.90 (t, 3H,J=7.0 Hz).

Synthesized by General Protocol A. 72% yield.

¹H-NMR (CDCl₃, 500 MHz): δ 5.72 (bs, 1H, major rotamer NH), 5.64 (bs,1H, minor rotamer NH), 3.37 (q, 2H, J=7.0 Hz, minor rotamer NCH₂), 3.26(q, 2H, J=7.0 Hz, major rotamer NCH₂), 2.01 (d, 3H, J=1.0 Hz, minorrotamer allylic CH₃), 1.93 (d, 3H, J=1.0 Hz, major rotamer allylic CH₃),1.50 (pent, 2H, J=7.0 Hz), 1.34-1.20 (m, 18H), 0.87 (t, 3H, J=7.0 Hz).¹³C-NMR (CDCl₃, 125 MHz): δ 153.61 (major), 82.73 (major), 75.05(major), 43.36 (minor), 39.81 (major), 31.92 (major), 30.55 (minor),29.66 (major), 29.64 (major), 29.61 (major), 29.56 (major), 29.37(major), 29.31 (2C, major), 26.90 (major), 26.53 (minor), 22.69 (major),14.12 (major), 3.95 (minor), 3.62 (major).

Synthesized by General Protocol C. 98% yield.

¹H-NMR (CDCl₃, 500 MHz): δ 6.22 (q, 1H, J=1.5 Hz, vinyl CH), 5.93 (bs,1H, NH), 3.29 (q, 2H, J=6.0 Hz), 2.62 (d, 3H, J=1.5 Hz, NCH₃), 1.52(pent, 2H, J=7.5 Hz), 1.35-1.20 (m, 18H), 0.85 (t, 3H, J=7.0 Hz).¹³C-NMR (CDCl₃, 125 MHz): δ 164.97, 129.73, 105.42, 39.74, 35.80, 32.07,29.80, 29.79, 29.74, 29.71, 29.59, 29.50, 29.46, 27.20, 22.84, 14.27.

Synthesized by General Protocols D, E, F, and H. 15% yield over 4 steps.

¹H-NMR (CDCl₃, 500 MHz): δ 10.3 (bs, 1H, NH), 6.75 (d, 1H, J=1.0 Hz,vinyl CH), 6.68 (s, 1H, vinyl CH), 4.48 (t, 2H, J=8.0 Hz, NCH₂), 2.61(d, 3H, J=0.5 Hz, allylic CH₃), 2.59 (d, 3H, J=1.0 Hz, allylic CH₃),1.69 (pent, 2H, J=7.5 Hz, NCH₂CH₂—), 1.42 (pent, 2H, J=7.5 Hz,NCH₂CH₂CH₂—), 1.38-1.18 (m, 16H), 0.86 (t, 3H, J=7.0 Hz, —CH₂CH₃).¹³C-NMR (CDCl₃, 125 MHz): δ 181.78, 175.27, 161.27, 130.99, 151.38,149.30, 139.13, 137.98, 128.64, 128.40, 119.69, 114.66, 46.50, 32.12,29.87, 29.85, 29,81, 29.80, 29.55, 29.44, 29.39, 27.14, 23.55, 22.83,22.44, 14.32. HRMS (ESI-TOF) calcd for C₂₆H₂₅N₂O₄ (M+H)⁺: 439.2597,found: 439.2595.

Synthesized by General Protocol A. 73% yield.

HRMS (ESI) calcd for C₉H₁₆NO (M+H)⁺: 154.1232, found: 154.1233.

Synthesized by General Protocol C. 95% yield.

¹H-NMR (CDCl₃, 500 MHz): δ 6.22 (q, 1H, J=1.5 Hz, vinyl CH), 5.82 (bs,1H, NH), 3.33 (dq, 2H, J=7.5, 1.0 Hz), 2.63 (d, 3H, J=1.5 Hz, allylicCH₃), 1.64 (sept, 1H, J=6.5 Hz), 1.43 (q, 2H, J=7.0 Hz), 0.91 (d, 6H,J=6.5 Hz). HRMS (ESI-TOF) calcd for C₉H₁₇NOI (M+H)⁺: 232.0355, found:282.0351.

Synthesized by General Protocols D, E, F, and H. 12% yield over 4 steps.

¹H-NMR (2:1 CDCl₃:CD₃OD, 500 MHz): δ 6.76 (q, 1H, J=1.0 Hz, vinyl CH),6.67 (q, 1H, J=1.5 Hz, vinyl CH), 4.52-4.49 (m, 2H), 2.64 (d, 3H, J=1.0Hz, allylic CH₃), 2.63 (d, 3H, J=1.0 Hz, allylic CH₃), 1.81 (sept, 1H,J=7.0 Hz, CH), 1.68-1.63 (m, 2H, CH2CH₂CH), 1.03 (d, 6H, J=6.5 Hz,CH(CH₃)₂). ¹³C-NMR(2:1 CDCl₃:CD₃OD, 125 MHz): δ 181.48, 175.02, 161.83,161.65, 151.69, 149.89, 139.30, 138.50, 127.42, 127.23, 119.52, 114.77,45.45, 37.28, 26.49, 22.89, 22.02, 21.83. HRMS (ESI-TOF) calcd forC₁₉H₂₁N₂O₄ (M+H)⁺: 341.1501. found: 341.1507.

Synthesized by General Protocol A. 25% yield.

¹H-NMR (CDCl₂, 500 MHz): δ 5.75 (bs, 1H, major rotamer NH), 3.17 (d, 2H,J=7.0 Hz, minor rotamer NCH₂), 3.09 (d, 2H, J=6.5 Hz, major rotamerNCH₂), 2.01 (s, 3H, minor rotamer allylic CH₃), 1.94 (s, 3H, majorrotamer allylic CH₃), 0.93 (s, 9H, minor rotamer), 0.92 (s, 9H, majorrotamer), HRMS (ESI) calcd for C₉H₁₆NO (M+H)⁺: 154.1232, found:154.1233.

Synthesized by General Protocol C. 96% yield.

¹H-NMR (CDCl₃, 500 MHz): δ 6.29 (q, 1H, J=1.5 Hz, vinyl CH), 5.78 (bs,1H, NH), 3.16 (d, 2H, J=6.0 Hz), 2.66 (d, 3H, J=1.0 Hz), 0.96 (s, 9H).HRMS (ESI-TOF) calcd for C₉H₁₇NOI (M+H)⁺: 282.0355, found: 282.0354.

Synthesized by General Protocols D, E, F, and H. 10% yield over 4 steps.

¹H-NMR (2:1 CDCl₃:CD₃OD, 500 MHz): δ 6.75 (d, 1H, J=1.0 Hz, vinyl CH),6.67 (d, 1H, J=1.0 Hz, vinyl CH), 4.95 (bs, 1H), 4.86 (bs, 1H), 2.64 (s,6H, allylic CH₃), 0.87 (s, 9H, (CH₃)₃). ¹³C-NMR (CDCl₃, 125 MHz): δ181.20, 176.32, 162.40, 149.55, 141.41, 139.05, 127.31, 127.19, 119.33,114.87, 51.10, 34.31, 27.58, 22.80, 21.67. HRMS (ESI-TOF) calcd forC₁₉H₂₁N₂O₄ (M+H)⁺: 341.1501, found: 341.1498.

Synthesized by General Protocol A. 76% yield.

¹H-NMR (CDCl₃, 500 MHz): δ 6.00 (bs, 1H, minor rotamer NH), 5.83 (bs,1H, major rotamer NH), 3.37-3.32 (m, 2H, minor rotamer NCH₂), 3.27-3.23(m, 2H, major rotamer NCH₂), 1.99 (s, 3H, minor rotamer allylic CH₃),1.89 (s, 3H, major rotamer allylic CH₃), 1.45-1.42 (m, 2H, minorrotamer), 1.41-1.38 (m, 2H, major rotamer), 0.91 (s, 9H, minor rotamer),0.89 (s, 9H, major rotamer), HRMS (ESI) calcd for C₁₀H₁₈NO (M+H)⁺:168.1388, found: 168.1387.

Synthesized by General Protocol C. 93% yield.

¹H-NMR (CDCl₃, 500 MHz): δ 6.20 (q, 1H, J=1.5 Hz, vinyl CH), 5.60 (bs,1H, NH), 3.37-3.33 (m, 2H), 2.65 (d, 3H, J=1.5 Hz, NCH₃), 1.48-1.45 (m,2H), 0.94 (s, 9H). HRMS (ESI-TOF) calcd for C₁₀H₁₉NOI (M+H)⁺: 296.0511,found: 296.0513.

Synthesized by General Protocols D, E, F, and H. 17% yield over 4 steps.

¹H-NMR (2:1 CDC₃:CD₃OD, 500 MHz): δ 6.75 (d, 1H, J=1.0 Hz, vinyl CH),6.66 (d, 1H, J=1.0 Hz, vinyl CH), 4.57-4.53 (m, 2H), 2.63 (d, 3H, J=1.0Hz, allylic CH₃), 2.62 (d, 3H, J=1.0 Hz, allylic CH₃), 1.66 (m, 2H,CH₂CH₂C(CH₃)₃, 1.07 (s, 9H, C(CH₃)₃). ¹³C-NMR (2:1 CDCl₃:CD₃OD, 125MHz): δ 181.46, 174.98, 161.81, 161.60, 151.64, 149.82, 139.35, 138.45,127.38, 127.21, 119.53, 114.74, 43.73, 41.45, 29.97, 28.87, 22.88,21.81. HRMS (ESI-TOF) calcd for C₂₀H₂₃N₂O₄ (M+H)⁺: 355.1658, found:355.1664.

Synthesized by General Protocol A. 65% yield.

¹H-NMR (CDCl₃, 500 MHz): δ 6.27 (bs, 1H, major rotamer NH), 6.02 (bs,1H, minor rotamer NH), 3.46 (t, 2H, J=6.0 Hz), 3.38 (q, 2H, J=6.5 Hz),3.34 (s, 3H), 1.92 (s, 3H), 1.77 (pent, 2H, J=6.0 Hz). ¹³C-NMR (CDCl₃,125 MHz): δ 156.37 (minor), 153.62 (major), 89.82 (minor), 82.95(major), 75.00 (major), 72.58 (minor), 71.28 (major), 70.45 (minor),58.77 (major), 41.34 (minor), 38.04 (major), 30.15 (minor), 28.87(major), 3.97 (minor), 3.64 (major).

Synthesized by General Protocol C. 96% yield.

¹H-NMR (CDCl₃, 500 MHz): δ 6.26 (bs, 1H, NH), 6.21 (d, 1H, J=1.0 Hz,vinyl CH), 3.49 (t, 2H, J=6.0 Hz), 3.42 (q, 2H, J=6.5 Hz), 3.34 (s, 3H),2.64 (d, 3H, J=1.5 Hz), 1.81 (pent, 2H, J=6.0 Hz). ¹³C-NMR (CDCl₃, 125MHz): δ 164.90, 129.40, 105.51, 71.65, 58.93, 38.02, 35.78, 29.07.

Synthesized by General Protocols D, E, G, and H.

¹H-NMR (2:1 CDCl₃:CD₃OD, 500 MHz): δ 6.76 (d, 1H, J=1.0 Hz, vinyl CH),6.65 (d, 1H, J=1.0 Hz, vinyl CH), 4.58 (t, 2H, J=7.5 Hz), 3.70 (t, 2H,J=6.0 Hz), 2.63 (d, 3H, J=1.0 Hz, allylic CH₂), 2.63 (d, 3H, J=1.0 Hz,allylic CH₃), 2.06-2.01 (m, 2H). HRMS (ESI-TOF) calcd for C₁₇H₁₇N₂O₅(M+H)⁺: 329.1137, found: 329.1129.

Molecular Modeling of DNQ in NQO1. DNQ (or derivative) was built and a10 Å water layer was built around the molecule. The DNQ structure wasthen energy minimized using MOE with a MMFF94x forcefield using gasphase calculations and a cutoff of 0.01. Charges were then fixed usingan MMFF94 forcefield. The NQO1 structure was downloaded from the PDB(2F1O). One of the homodimers was extracted and protonated. DNQ was thenmodeled into the protein active site, using the site of dicoumarol toidentify the active site. It was docked using the Dock program in MOEwhich uses Triangle Matching for the placement of the small molecule andLondon dG for rescoring of the placement of the small molecule. The top30 configurations were then visually inspected to ensure that themolecule was within the active site and pi stacking with the FADmolecule. Using LigX, the best configuration was protonated and theenergy was minimized to obtain the calculated binding energies.

Example 5 Inhibition of Breast Cancer Cells

FIG. 12 illustrates an embodiment of the efficacy of DNQ and compound 87vs. the MDA-MB-231 (breast cancer) cell line that expresses NQO1, theversion that does not express NQO1, and both these cell lines where NQO1is inhibited by dicumoral.

Example 6 DNQ Derivative PAR-PARP1 Formation in LLC Tumors in Vivo

Immunoblotting and Antibodies: Antibodies that specifically detectpoly(ADP-ribosyl)ated α-PAR (BD Pharmingen, San Jose, Calif.) proteins(e.g., the most abundant species, poly(ADP-ribosyl)ated PARP1,PAR-PARP1) and α-PARP1 (sc-8007, Santa Cruz Biotechnology) antibodieswere used at 1:4000 and 1:2000 dilutions, respectively. α-Tubulin wasmonitored for loading.

PARP1 hyperactivation in vivo Using the Orthotopic Lewis Lung Carcinoma(LLC) model: To generate a known control to monitor the formation ofpoly(ADP-ribose)ylated-polymerase 1 (PAR-PARP1) in cells, LLC tumorcells were treated in vitro with hydrogen peroxide (H₂O₂, 0.5 μM) for 15minutes, and whole cell protein lysates were separated by SDS-PAGE usingthe α-PAR (BD Pharmingen, San Jose, Calif.) antibody described above.PAR-PARP1 formation was then assessed by immunoblot analyses aspreviously described in Huang et al., Cancer Research, 2012.

LLC cells (1.0×10⁶) were intravenously (iv) injected into the tail veinsof female 18-20 gram NOD/SCID mice. Establishment of tumor nodules inthe lungs of exposed mice were confirmed by bioluminescence and mice(5/group) were treated intravenously (via tail vein injection) with: (i)hydroxypropyl-beta-cyclodextrin (HPβCD) vehicle alone; (iii) DNQ (7.5mg/kg); (ii) DNQ-87 (14 mg/kg); (iii) DNQ-107 (1.0 mg/kg); or (vi) DNQ9-251 (20 mg/kg) dissolved in HPβCD. At specific times indicated (inmins) after injection of vehicle or drug, mice were euthanized, lungsremoved and tumor nodules harvested. Whole cell extracts were preparedfrom rumor tissue and formation of PAR-PARP1 was evaluated byimmunoblotting analyses. Extracts from cells treated with H₂O₂ were usedas controls for monitoring PAR-PARP1 formation in LLC tumor cells. FIG.13 shows the immunoblotting analyses of DNQ-87, DNQ-107 and DNQ-9-251.

Results: Using an orthotopic LLC model in female NOD/SCID mice, PARP1hyperactivation in vivo was monitored by the formation of PAR-PARP1after exposure to DNQ, DNQ-87, DNQ-107, or DNQ 9-251. Each DNQ drug wasused at or near their maximum tolerated doses (MTDs). Analyses of tumortissue from exposed mice revealed the formation of PAR-PARP1 followingtreatment with DNQ, while tumors treated with vehicle alone did notdisplay PARP1 hyperactivation. Additionally, the treatment oftumor-bearing animals with DNQ-87, DNQ-107 or DNQ 9-251 not onlyresulted in formation of PAR-PARP1, but higher levels of PAR-PARP1formation were noted than in mice treated with DNQ.

In vitro, DNQ-87 was also an efficient inducer of specific types of DNAdamage. Like DNQ and β-lapachone, DNQ-87 caused extensive initial DNAsingle strand damage and base lesions as measured by alkaline cometassays (data not shown). In contrast, DNA double strand break (DSB)formation was delayed (appearing 15-30 mins after initial DNA SSBs andbase lesions), because DNQ-87-exposed cells demonstrated increases innuclear staining of phosphorylated (Ser-1981) ATM, γ-H2AX andphosphorylated DNA-PKcs proteins that are hallmarks of exposed DSBs(data not shown). Thus, DNQ-87, like DNQ and β-lapachone, are efficientDNA base and SSB lesions formation, with delayed formation of DSBs mostlikely due to DNA replication.

Example 7 Preparation of DNQ Compounds

various DNQ compounds, such as DNQ-P1, DNQ-P2, DNQ-P3, DNQ-P4, andvarious other derivatives, can be prepared by the methods illustrated inSchemes 7.1 to 7.4. The compounds can be isolated as either thephosphoric acids or, for example, as the corresponding sodium salts. Thephosphate compounds can have significantly higher aqueous solubilitythen the corresponding alkyl compounds.

Cross coupling, ring closure, deprotection with HBr and oxidation canthen be carried out as shown in Scheme 1.14 and/or as described inExample 4 to provide DNQ-6.2a or DNQ-6.2b, which can then be modified toprovide DNQ-P2 or DNQ-P3, respectively, as shown in Scheme 7.3.

The final intermediate of Scheme 7.4 can be converted to DNQ-P4 by aroute similar to that employed in Scheme 7.3 to provide thephosphorylated product, which can be readily isolated as a sodium salt.

Example 8 Pharmaceutical Dosage Forms

The following formulations illustrate representative pharmaceuticaldosage forms that may be used for the therapeutic or prophylacticadministration of a compound of a formula described herein, a compoundspecifically disclosed herein, or a pharmaceutically acceptable salt orsolvate thereof (hereinafter referred to as ‘Compound X’):

(i) Tablet 1 mg/tablet ‘Compound X’ 10.0 Lactose 77.5 Povidone 15.0Croscarmellose sodium 12.0 Microcrystalline cellulose 92.5 Magnesiumstearate 3.0 210.0

(ii) Tablet 2 mg/tablet ‘Compound X’ 20.0 Microcrystalline cellulose410.0 Starch 50.0 Sodium starch glycolate 15.0 Magnesium stearate 5.0500.0

(iii) Capsule mg/capsule ‘Compound X’ 10.0 Colloidal silicon dioxide 1.5Lactose 465.5 Pregelatinized starch 120.0 Magnesium stearate 3.0 600.0

(iv) Injection 1 (1 mg/mL) mg/mL ‘Compound X’ (free acid form) 1.0Dibasic sodium phosphate 12.0 Monobasic sodium phosphate 0.7 Sodiumchloride 4.5 1.0N Sodium hydroxide solution q.s. (pH adjustment to7.0-7.5) Water for injection q.s. ad 1 mL

(v) Injection 2 (10 mg/mL) mg/mL ‘Compound X’ (free acid form) 10.0Monobasic sodium phosphate 0.3 Dibasic sodium phosphate 1.1 Polyethyleneglycol 400 200.0 0.1N Sodium hydroxide solution q.s. (pH adjustment to7.0-7.5) Water for injection q.s. ad 1 mL

(vi) Aerosol mg/can ‘Compound X’ 20 Oleic acid 10Trichloromonofluoromethane 5,000 Dichlorodifluoromethane 10,000Dichlorotetrafluoroethane 5,000

(vii) Topical Gel 1 wt. % ‘Compound X’   5% Carbomer 934 1.25%Triethanolamine q.s. (pH adjustment to 5-7) Methyl paraben  0.2%Purified water q.s. to 100 g

(viii) Topical Gel 2 wt. % ‘Compound X’ 5% Methylcellulose 2% Methylparaben 0.2%  Propyl paraben 0.02%   Purified water q.s. to 100 g

(ix) Topical Ointment wt. % ‘Compound X’ 5% Propylene glycol 1%Anhydrous ointment base 40%  Polysorbate 80 2% Methyl paraben 0.2% Purified water q.s. to 100 g

(x) Topical Cream 1 wt. % ‘Compound X’  5% White bees wax 10% Liquidparaffin 30% Benzyl alcohol  5% Purified water q.s. to 100 g

(xi) Topical Cream 2 wt. % ‘Compound X’ 5% Stearic acid 10%  Glycerylmonostearate 3% Polyoxyethylene stearyl ether 3% Sorbitol 5% Isopropylpalmitate 2% Methyl Paraben 0.2%  Purified water q.s. to 100 g

These formulations may be prepared by conventional procedures well knownin the pharmaceutical art. It will be appreciated that the abovepharmaceutical compositions may be varied according to well-knownpharmaceutical techniques to accommodate differing amounts and types ofactive ingredient ‘Compound X’. Aerosol formulation (vi) may be used inconjunction with a standard, metered dose aerosol dispenser.Additionally, the specific ingredients and proportions are forillustrative purposes. Ingredients may be exchanged for suitableequivalents and proportions may be varied, according to the desiredproperties of the dosage form of interest.

The terms and expressions which have been employed herein are used asterms of description and not of limitation, and there is no intention inthe use of such terms and expressions of excluding any equivalents ofthe features shown and described or portions thereof, but it isrecognized that various modifications are possible within the scope ofthe invention claimed. Thus, it should be understood that although thepresent invention has been specifically disclosed by preferredembodiments, exemplary embodiments and optional features, modificationand variation of the concepts herein disclosed may be resorted to bythose skilled in the art, and that such modifications and variations areconsidered to be within the scope of this invention as defined by theappended claims. The specific embodiments provided herein are examplesof useful embodiments of the present invention and it will be apparentto one skilled in the art that the present invention may be carried outusing a large number of variations of the devices, device components,methods steps set forth in the present description. As will berecognized by one of skill in the art, methods and devices useful forthe present methods can include a large number of optional compositionand processing elements and steps.

Although the present invention has been described with reference tocertain embodiments thereof, other embodiments are possible withoutdeparting from the present invention. Although the description hereincontains a plurality of specificities, these should not be construed aslimiting the scope of the invention but as merely providingillustrations of some of the presently preferred embodiments of theinvention. The spirit and scope of the appended claims should not belimited, therefore, to the description of any specific embodimentscontained herein. All embodiments that come within the meaning of theclaims, either literally or by equivalence are intended to be embracedtherein. Furthermore, the advantages described above are not necessarilythe only advantages of the invention, and it is not necessarily expectedthat all of the described advantages will be achieved with everyembodiment of the invention.

All references throughout this application, for example patent documentsincluding issued or granted patents or equivalents; patent applicationpublications; and non-patent literature documents or other sourcematerial; are hereby incorporated by reference herein in theirentireties, as though individually incorporated by reference, to theextent each reference is at least partially not inconsistent with thedisclosure in this application (for example, a reference that ispartially inconsistent is incorporated by reference except for thepartially inconsistent portion of the reference). References citedherein are incorporated by reference to indicate the state of the art asof their publication or filing date and it is intended that thisinformation can be employed herein, if needed, to exclude specificembodiments that are in the prior art. For example, when compounds areclaimed generically, it should be understood that compounds known andavailable in the art prior to Applicant's invention, including compoundsfor which an enabling disclosure is provided in the references citedherein, are not intended to be included in the compounds claims herein.

In view of the many possible embodiments to which the principles of thedisclosure may be applied, it should be recognized that the illustratedembodiments are only examples of the disclosure and should not be takenas limiting the scope of the invention. Rather, the scope of thedisclosure is defined by the following claims. We therefore claim as ourinvention all that comes within the scope and spirit of these claims.

What is claimed is:
 1. A compound of the Formula (I):

wherein R₁, R₂, R₃, and R₄ are each independently —H or —X—R; each X isindependently a direct bond or a bridging group, wherein the bridginggroup is —O—, —S—, —NH—, —C(═O)—, —O—C(═O)—, —C(═O)—O—, —O—C(═O)—O—, ora linker of the formula -W-A-W-, wherein each W is independently—N(R′)C(═O)—, —C(═O)N(R′)—, —OC(═O)—, —C(═O)O—, —O—, —S—, —S(O)—,—S(O)₂—, —N(R′)—, —(═O)—, —(CH₂)_(n)— where n is 1-10, or a direct bond,wherein each R′ is independently H, (C₁-C₆)alkyl, or a nitrogenprotecting group; and each A is independently (C₁-C₂₀)alkyl,(C₂-C₁₆)alkenyl, (C₂-C₁₆)alkynyl, (C₃-C₈)cycloalkyl, (C₆-C₁₀)aryl,—(OCH₂—CH₂)_(n)— where n is 1 to about 20, —C(O)NH(CH₂)_(n)— wherein nis 1 to about 6, —OP(O)(OH)O—, —OP(O)(OH)O(CH₂)_(n)— wherein n is 1 toabout 6, or (C₁-C₂₀)alkyl, (C₂-C₁₆)alkenyl, (C₂-C₁₆)alkynyl, or—(OCH₂—CH₂)_(n)— interrupted between two carbons, or between a carbonand an oxygen, with a cycloalkyl, heterocycle, or aryl group; each R isindependently alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl,cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, (cycloalkyl)alkyl,(heterocycloalkyl)alkyl, (cycloalkyl)heteroalkyl,(heterocycloalkyl)heteroalkyl, aryl, heteroaryl, (aryl)alkyl,(heteroaryl)alkyl, hydrogen, hydroxy, hydroxyalkyl, alkoxy,(alkoxy)alkyl, alkenyloxy, alkynyloxy, (cycloalkyl)alkoxy,heterocycloalkyloxy, amino, alkylamino, aminoalkyl, acylamino,arylamino, sulfonylamino, sulfinylamino, —COR^(x), —COOR^(x),—CONHR^(x), —NHCOR^(x), —NHCOOR^(x), —NHCONHR^(x), —N₃, —CN, —NC, —NCO,—NO₂, —SH, -halo, alkoxycarbonyl, alkylaminocarbonyl, sulfonate,sulfonic acid, alkylsulfonyl, alkylsulfinyl, arylsulfonyl, arylsulfinyl,aminosulfonyl, R^(x)S(O)R^(y)—, R^(x)S(O)₂R^(y)—,R^(x)C(O)N(R^(x))R^(y)—, R^(x)SO₂N(R^(x))R^(y)—,R^(x)N(R^(x))C(O)R^(y)—, R^(x)N(R^(x))SO₂R^(y)—,R^(x)N(R^(x))C(O)N(R^(x))R^(y)—, carboxaldehyde, acyl, acyloxy, —OPO₃H₂,—OPO₃Z₂ where Z is an inorganic cation, or saccharide; where each R^(x)is independently H, OH, alkyl or aryl, and each R^(y) is independently agroup W; wherein any alkyl or aryl can be optionally substituted withone or more hydroxy, amino, cyano, nitro, or halo groups; or a salt orsolvate thereof; provided that when R₁, R₂, and R₃ are methyl, R₄ is notH or methyl; and provided that when R₁, R₃, and R₄ are methyl, the group—X—R of R₂ is not acyloxy.